US20150080256A1 - Luminescence detecting apparatuses and methods - Google Patents
Luminescence detecting apparatuses and methods Download PDFInfo
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- US20150080256A1 US20150080256A1 US14/515,433 US201414515433A US2015080256A1 US 20150080256 A1 US20150080256 A1 US 20150080256A1 US 201414515433 A US201414515433 A US 201414515433A US 2015080256 A1 US2015080256 A1 US 2015080256A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N21/15—Preventing contamination of the components of the optical system or obstruction of the light path
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
- G01N21/274—Calibration, base line adjustment, drift correction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
- G01N21/274—Calibration, base line adjustment, drift correction
- G01N21/276—Calibration, base line adjustment, drift correction with alternation of sample and standard in optical path
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
- G01N2021/6471—Special filters, filter wheel
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/11—Automated chemical analysis
- Y10T436/115831—Condition or time responsive
Definitions
- This invention relates to the field of apparatus and methods for detecting and quantifying light emissions, and more particularly, to detecting and quantifying light emitted from luminescent-based assays. Still more particularly, this invention pertains to apparatus and methods for detecting and quantifying luminescence such as bioluminescence and/or chemiluminescence from luminescent assays as an indicator of the presence or amount of a target compound.
- Preferred embodiments of the invention include as an imaging device a charge coupled device (CCD) camera and a computer for analyzing data collected by the imaging device. Further preferred embodiments include the capacity for use in high throughput screening (HTS) applications, and provide for robot handling of assay plates.
- CCD charge coupled device
- HTS high throughput screening
- Such luminescence immunoassays offer the potential of combining the reaction specificity of immunospecific antibodies or hybridizing nucleic acid sequences and similar specific ligands with the high sensitivity available through light detection.
- radioactive reagents have been used for such purposes, and the specificity and sensitivity of LIA reagents is generally comparable to those employing traditional radiolabelling.
- LIA is the preferred analytical method for many applications, owing to the nontoxic nature of LIA reagents and the longer shelf lives of LIA reagents relative to radioactive reagents.
- chemiluminescent compounds such as 1,2-dioxetanes, developed by Tropix, Inc. and other stable chemiluminescent molecules, such as xanthan esters and the like, are in commercial use. These compounds are triggered to release light through decomposition triggered by an agent, frequently an enzyme such as alkaline phosphatase, which is present only in the presence, or specific absence, of the target compound. The detection of light emission is a qualitative indication, and the amount of light emitted can be quantified as an indicator of the amount of triggering agent, and therefore target compound, present.
- Other well known luminescent compounds can be used as well.
- Luminescent release may sometimes be enhanced by the presence of an enhancement agent that amplifies or increases the amount of light released. This can be achieved by using agents which sequester the luminescent reagents in a microenvironment which reduces suppression of light emission. Much biological work is done, perforce, in aqueous media. Water typically suppresses light emission. By providing compounds, such as water soluble polymeric onium salts (ammonium, phosphonium, sulfonium, etc.) small regions where water is excluded that may sequester the light emitting compound may be provided.
- an enhancement agent that amplifies or increases the amount of light released. This can be achieved by using agents which sequester the luminescent reagents in a microenvironment which reduces suppression of light emission. Much biological work is done, perforce, in aqueous media. Water typically suppresses light emission.
- compounds such as water soluble polymeric onium salts (ammonium, phosphonium, sulfonium, etc.) small regions where water is excluded that
- the majority of instrumentation used to monitor light emitting reactions use one or more photomultiplier tubes (PMTs) to detect the photons emitted. These are designed to detect light at the low light levels associated with luminescent reactions.
- PMTs photomultiplier tubes
- the rate at which a PMT based microplate luminometer can measure signal from all wells of the plate is limited by the number of PMTs used. Most microplate luminometers have only one PMT so a 384 well plate requires four times longer than is required to read a 96-well plate.
- U.S. Pat. No. 4,772,453 describes a luminometer having a fixed photodetector positioned above a platform carrying a plurality of sample cells. Each cell is positioned in turn under an aperture through which light from the sample is directed to the photodetector.
- U.S. Pat. No. 4,366,118 describes a luminometer in which light emitted from a linear array of samples is detected laterally instead of above the sample.
- EP 0025350 describes a luminometer in which light emitted through the bottom of a sample well is detected by a movable photodetector array positioned underneath the wells.
- a variety of light detection systems for HTS applications are available in the market. These include the LEADseekerTM from Amersham/Pharmacia, the ViewLuxTM offered by PerkinElmer and CLIPRTM from Molecular Devices. These devices are all expensive, large dimensioned (floorbased models), exhibit only limited compatibility with robotic devices for plate preparation and loading, have a limited dynamic range, and/or use optical detection methods which do not reduce, or account for, crosstalk.
- the optical systems used are typically complex teleconcentric glass lens systems, which may provide a distorted view of wells at the edges of the plates, and the systems are frequently expensive, costing in excess of $200,000.00.
- Perhaps the most popular detection apparatus is the TopCountTM, a PMT-based detection system from Packard. Although the TopCountTM device has a desirable dynamic range, it is not capable of reading 1,536 well plates, and it does not image the whole plate simultaneously.
- Crosstalk from adjacent samples remains a significant obstacle to the development of improved luminescence analysis in imaging-based systems. This can be appreciated as a phenomenon of simple optics, where luminescent samples produce stray light which can interfere with the light from adjacent samples. Furthermore, the development of luminometers capable of detecting and analyzing samples with extremely low light levels are particularly vulnerable to crosstalk interference.
- a luminescence detecting apparatus that will permit the analysis of luminescent samples. It is a further object of the present invention to provide a luminescence detecting apparatus capable of simultaneously analyzing a large number of luminescent samples.
- a luminescence detecting apparatus is provided that simultaneously analyzes multiple samples held in wells, where the well plates contain as many as 1,536 wells.
- the present invention further includes robot handling of the multiple well trays during analysis.
- the apparatus of this invention employs a Fresnel lens arrangement, with a vertical collimator above the well plate, with dimensions to match the number of wells.
- a 1,536-well plate will employ a dark collimator above the plate with 1,536 cells in registry with the wells of the plate.
- Fixed above the collimator is a Fresnel lens, which refracts the light such that the view above the lens appears to be looking straight down into each well, regardless of its position on the plate, even at the edges.
- the Fresnel lens is a CCD camera arranged so as to take the image of the entire plate at one time, viewing through a 35 mm wide angle lens, to give whole plate imaging on a rapid basis.
- a filter typically arrayed on a filter wheel, disposed at an angle to the lens.
- the filter is selected to permit the passage of the specific wavelength of the light emitted, and reflect or absorb all others.
- filters may be provided on the wheel, to permit sequential detection of light emitted from multiple reagents emitting light at different wavelengths.
- the samples are fed to the optical detection platform through a loading device designed to work well with robotic and automated preparation systems.
- the well-plate, with reaction mixture already provided, is placed on a shuttle by a human, or preferably, robot. Alignment of the plate on the shuttle may be relatively coarse, notwithstanding the requirement for tight tolerances to match the collimator grid array.
- a resilient means urges the plate into strict conformal alignment.
- the shuttle positions the plate under an overhead injection bar, which may accommodate up to sixteen wells in a column at one time. If not previously added, a triggering agent or luminescent reagent is added to the sample wells, and the plate indexes forward to load the next column of wells across the plate.
- the shuttle then advances through a door into the sample chamber, and the plate is aligned with the collimator and the Fresnel lens. Since many reactions proceed better, or only, at elevated temperatures, the sample chamber is insulated, and provided with heating means, for heating the air in or provided to the chamber. In order to maintain temperature in the chamber close to room temperature and to accurately control temperature, the chamber may also be provided with a heat exchanger.
- the light emission from the entire multiple well plate is imaged at once, with subsequent imaging through a different filter if multiple wavelengths are employed.
- the signal obtained is processed to further reduce crosstalk reduced by the collimator and the presence and amount of luminescence is quickly detected and calculated by a personal computer using automated software. Data is then reported as intensity per well or further analyzed relative to specific assay standards.
- FIG. 1 is a cross section of a preferred embodiment of a luminescence detecting apparatus according to the present invention
- FIG. 2 is a detailed cross section of the optics of a luminescence detecting apparatus according to the present invention.
- FIG. 3 is a cross-sectional view of the plate transport system of the invention.
- FIG. 4 is a perspective illustration of the injector arm assembly of the invention.
- FIG. 5 is an exploded view of the filter wheel assembly.
- FIG. 6 is a cross-sectional view of the optical housing.
- FIG. 6A is a plan view of a robotic mechanism of the invention.
- FIG. 7 is a flow chart illustration of the processing method of the invention.
- FIGS. 8-15 are illustrations of the results obtained using the invention in Examples 1-10, respectively.
- a preferred embodiment of the luminescence detecting apparatus of the present invention uses a shuttle or tray to carry a micro plate (plate) 10 comprising a plurality of sample wells 20 which may in the preferred embodiment number as many as 1,536 or more.
- a micro plate plate 10 comprising a plurality of sample wells 20 which may in the preferred embodiment number as many as 1,536 or more.
- the sample wells 20 may be filled with analyte manually, or robotically prior to delivery to the inventive apparatus.
- Agents necessary for chemiluminescence may be filled automatically via the injector 30 , to which analyte is supplied through an array of supply tubes 40 or prior to placing the plate on the tray.
- the sample wells will contain chemiluminescent reagents. These reagents emit light at intensities proportional to the concentration of analyte in the sample. This light can be very low intensity and requires an instrument with sufficient sensitivity to achieve the desired detection limits.
- injector 30 is controlled by central processor 50 , which in the preferred embodiment may control the operation of all elements of the luminometer of the present invention. Data collection, analysis and presentation may also be controlled by processor 50 . Further in a preferred embodiment of the present invention, the injector 30 may also be used to add buffer solutions to the analytes and also to add reagents that enable “glow” and/or “flash” luminescence imaging, that is sustained or brief, intense emission, respectively, all under control of central processor 50 .
- sample chamber 55 which is located in optical chamber 60 at a fixed focal distance from and directly under the charge-coupled device (CCD) camera 70 , in order to permit the CCD camera to image the luminescent sample accurately.
- the sample chamber 55 is preferably capable of precise temperature control, as many luminescent reagents and specific luminescent reactions are temperature dependent. Temperature control is provided by central processor 50 , which can vary the temperature for each individual sample plate 10 , as central processor 50 controls the movement and injection of the sample wells 20 in each sample tray 10 . In a preferred embodiment of the luminometer of the present invention, central processor 50 also controls an industrial robot (not shown) which performs the activities involving analyte handling in the luminometer of the present invention.
- the optics 80 deliver the image of the complete microplate 10 as a single image to the CCD camera 70 .
- the operation of the luminometer of this invention is an integral, continuous practice, and all elements of the luminometer cooperate together to provide precise, accurate and reliable data
- the invention may be more easily understood by reference to three separate, integrated systems, the optics system, the mechanical system and the processing system. Each is discussed in turn, with a discussion of examples of the operation as a whole to follow.
- Luminescent emission 100 from the analyte in plate well 20 located in the plate 10 travels first through dark collimator 110 , which permits only parallel and semi-parallel light rays to exit the sample wells 20 for eventual imaging by the CCD camera 70 .
- the effect of collimation assists with the prevention of stray light from the sample wells 20 and with the elimination of crosstalk between luminescent samples.
- the collimator 110 may be sealably engaged, or in close proximity, to the sample tray 10 , to enhance the restriction of stray light from the samples.
- Each well 10 is in strict registration and alignment with a corresponding grid opening in collimator 110 .
- the luminescent radiation passes through a Fresnel field lens 120 , which focuses the light toward filter 130 .
- the collimator 110 and Fresnel field lens 120 are packaged in a cassette that can be changed by the user. Such an equipment change may be necessitated by varying optical characteristics of different analytes and different well distributions in plates.
- Fresnel field lens is preferable to alternative optical devices for several reasons. Initially, improvements in design and materials have capitalized on the superior optical capabilities of the Fresnel lens, while virtually eliminating its once inherent limitations. Today, many Fresnel lenses are made of molded plastic, creating an almost flawless surface with very little scatter light. The elimination of scatter light is an important element of eliminating crosstalk between adjacent samples in the luminometer of the present invention. Furthermore, improved types of plastics commonly employed in the manufacturing of Fresnel lenses and other optical devices have optical qualities equivalent to ground glass lenses.
- Fresnel lenses can be manufactured to produce the precise optical imaging effect that is most efficient for a charge coupled device camera, as in the present invention.
- Fresnel lenses offer an advantage over conventional lenses in that they can be molded flat and very thin. Because of the shape of the Fresnel lens, it can easily be integrated directly into the housing of the luminometer, enhancing the light-tight properties necessary for accurate imaging of low light samples. Furthermore, Fresnel lenses are much less expensive than comparable conventional glass lenses.
- the total beam spread from a Fresnel lens depends on the size of the source in relation to the focal length of the lens. Smaller sources, such as luminescent assay samples, and longer focal lengths produce more compact beams. Since there are practical limitations to minimizing the geometry and dimensions of the optics 80 in the luminometer of the present invention, the use of Fresnel field lens 120 provides the greatest opportunity for fine-tuned optics.
- the emissions from plate 10 pass through lens 120 , and are refracted such that the image obtained at CCD 70 appears to look directly downward into all wells, even laterally displaced (edge) ones. This feature is typically called “telecentric.”
- the filter 130 may be configured on a wheel, wherein different filter elements may occupy different portions of the wheel, depending on the luminescent characteristics of the sample being analyzed.
- Filter 130 is preferably inclined at an angle of 20°-30° relative to the CCD, so that stray reflected light is reflected outside the field of view.
- the filter wheel 130 permits the selection of different wavelength ranges, which not only permit high quality imaging, but may be used to separate the emissions of different reagents emitting at different wavelengths.
- the filter wheel 130 is controlled by central processor 50 , in coordination with central processor 50 's control of the individual sample wells 20 in the sample plate 10 . In many assays, such as those addressed in pending U.S. patent application Ser.
- Filter 130 is preferably provided with an infrared (IR) filter operating in conjunction with the selected bandpass, or as an independent element. Applicants have discovered that stray IR radiation, resulting from the plate phosphorescence, resulting in abnormally high backgrounds. An IR filter suppresses this.
- IR infrared
- CCD camera 70 is a cooled, low noise, high resolution device.
- the lens is preferably a 35 mm wide angle lens with a low light level (F1.4) large aperture character. Magnification of 3-6, preferably about 5.5, is preferred.
- CCD camera 70 is provided with an anti-blooming CCD chip, to enhance dynamic range, which is about 10 5 in the claimed invention, referred to as the NorthStarTM luminometer.
- the selected CCD camera includes a liquid cooled thermoelectric (Peltier) device providing cooling of the CCD to approximately ⁇ 35° C., and the CCD has 1280 ⁇ 1024 pixels, each of which are 16 ⁇ m square, producing a total active area of 20.5 mm ⁇ 16.4 mm.
- the quantum efficiency averages 15% over the range from 450 nanometers to 800 nm.
- the output is digitized to 16 bit precision and pixels can be “binned” to reduce electronic noise.
- the luminometer of the present invention has a spatial resolution capable of providing high quality imaging of high density, sample trays.
- the noise performance and CCD temperature are designed to provide the desired detection limit.
- the mechanical systems of the luminometer workstation of this invention are designed to achieve automated, high throughput precise delivery of microplates in registration with a collimator 110 so as to be read by the CCD Camera 70 .
- a cross-section of the inventive luminometer shuttle 200 translates from a load position 202 , where plates 10 are loaded on to the shuttle, preferably by a robotic device such as robot arm, and the shuttle 200 then translates towards sample chamber 55 , to read position 203 .
- Shuttle 200 is caused to translate by a conventional stepper motor (not pictured). As shuttle 200 advances toward sample chamber 55 , it may stop underneath injector 30 . Injector 30 is more fully illustrated below in FIG. 4 . Referring still to FIG.
- injector 30 delivers fluid reagents drawn from reservoir 204 .
- Syringe pump 205 draws the fluid reagents from reservoir 204 , and pumps the fluid to the injector tubes 40 .
- Two way valve 206 controls the passage of the fluid drawn by syringe pump 205 from reservoir 204 and pumped by syringe pump 205 to the supply tubes 40 .
- injector 30 has up to sixteen injection ports 302 .
- the plates used in conjunction with the luminometer when injection is used are typically prepared with up to sixteen wells in a column.
- shuttle 200 advances plate 10 underneath injector 30 .
- shuttle 200 stops so that the first column 208 of wells is directly aligned under injector 30 .
- Precise amounts of analyte are delivered to the first set of wells, and shuttle 200 indexes forward one column, so as to inject reagent into the second column of wells 210 . This process is repeated until all wells are filled.
- shuttle 200 advances forward into sample chamber 55 through hinged door 212 .
- door 212 may be a guillotine door or similar type of closing mechanism.
- the wells of plate 10 are then read in sample chamber 55 .
- shuttle 200 translates back to load position 202 .
- Trough 304 swings out from its storage position parallel to the direction of travel of shuttle 200 , shown by an arrow, to a position directly underlying the injector 30 , perpendicular to the direction of travel. Fluid in the injector and tubes 204 are delivered into trough 304 , and removed by suction. Trough 304 then returns to its rest position, parallel to, and away from, the direction of travel of the shuttle 200 , when the shuttle is moved toward the sample chamber 55 . On its return trip to load position 202 , locator 214 on shuttle 200 is engaged by cam 216 .
- Locator 214 is mounted on a resilient means, such that when engaged by cam 216 , the locator 214 recesses away from plate 10 . This permits removal of plate 10 , and delivery from a robotic arm or other source of a fresh plate 10 , without the requirement of precise location. As shuttle 200 moves away from load 202 , locator 214 is urged forward, firmly locating plate 10 in place. Plate 10 is held against shoulder 217 by the resilient urging of locator 214 .
- a bar code reader 218 is mounted on the luminometer housing generally indicated at 299 and directly above the door 212 , for example on an arm or flange 220 . Bar code reader 218 is focused on a mirror 222 which in turn permits reading directly off the front or leading edge of plate 10 as it approaches on shuttle 200 .
- processor 50 the results obtained can be correlated therewith.
- injector 30 may be precisely located by operation of actuator wheel 306 , provided with positions corresponding to the total number of wells on the plates being assayed. Similarly, the vertical position, to account for the different thicknesses of the plate, may be controlled by wheel 308 . Given the simple translation movement of shuttle 200 , and the precise locating and identification of each plate carried, rapid cycling of micro-plate test plates into and out of sample chamber 55 can be effected.
- a filter which includes or reflects passage of light other than light falling within the selected wavelength of the luminescent emitter in use.
- the filter assembly is illustrated in exploded format in FIG. 5 .
- Filter frame 502 is supported by arm 504 which is connected to the hub of the filter wheel 506 .
- Multiple different filters may be provided on a single wheel.
- the filter itself, 508 is securely mounted on the frame and held there by cover 510 , which is secured to frame 502 by grommets, screws or other holding devices 512 .
- filter wheel is positioned so as to hold filter 508 in frame 502 at in incline with respect to collimator 110 , of about 22° nominally, so as to direct any reflections outside the field of view.
- Light passes through the filter opening 514 , in alignment with camera lens 140 and CCD camera 70 .
- filter 508 preferably includes an infrared block, either as a component of the filter itself, or as a component provided in addition to the filter for the measured light.
- An IR block is of value to prevent infrared emissions caused by extraneous radiation from altering the image received by the CCD camera.
- Optical chamber 60 is more fully illustrated in FIG. 6 .
- optical chamber 60 is bounded by optical housing 602 in which fits sample housing 604 .
- sample housing 604 When a plate 10 is loaded into optical chamber 60 , the plate is secured in sample housing 604 which is positioned in registry with collimator 110 , over which is provided Fresnel lens 120 . While many luminescent assays can be provided at ambient temperatures, some require elevated temperatures.
- the luminometer of this device is provided with a sample chamber in which the sample housing 604 carries insulation 606 which, in a preferred embodiment is polyurethane foam, and heater element 608 to raise the temperature in the sample chamber 55 above ambient temperature, up to about 42° C.
- the defogger 610 directs a stream of air heated just a few degrees, preferably about 2-3° degrees, above ambient conditions, or above the temperature of the chamber if the chamber is above ambient conditions, across the surface of the Fresnel lens 120 , effectively preventing condensation.
- filter motor 610 mounted at the top of the interior of optical chamber 60 is filter motor 610 which drives filter wheel 612 , on which may be mounted filters 614 of varying wavelength, for filtering undesirable wavelengths prior to imaging.
- optical chamber 60 a region is provided, indicated at 616 , in the optical housing 602 of the optical chamber 60 for light to be directed onto the CCD camera after passing through the filter 614 .
- the dimensions of optical chamber 60 are exaggerated in FIG. 6 to illustrate the relationship between the optical chamber 60 and the filter wheel 612 , and defogger 610 .
- the filter is located inside the optical chamber 60 , and outside the sample housing 604 but alternate locations are possible while still achieving the desired function.
- FIG. 6A a plan view of a novel robotic mechanism 616 is displayed in a preferred embodiment of the present invention, which provides capacity for use in high throughput screening (HTS) applications.
- HTS high throughput screening
- robot plate stacks 620 , 622 , 624 , 626 , and 628 each can be filled with multiple sample plates 10 , arranged in a vertical stack.
- robot plate stack 628 is designated as the discard stack.
- the remaining robot plate stacks 620 , 622 , 624 , and 626 can be programmed in order of delivery by software controlled by processor 50 (not shown).
- processor 50 not shown
- robot arm 630 moves vertically and rotationally to the desired robot plate stack, under control of the software programmed in processor 50 .
- transport 200 of the instrument When commanded by processor 50 , transport 200 of the instrument will move the sample plate 10 from load position 202 to the Read position 203 , and return it to load position 202 when imaging is complete.
- the elapsed time between moving the sample plate 10 from load position 202 to the read position 203 , and returning it to load position 202 is typically 30-120 seconds, including imaging time.
- Staging positions 632 and 634 are located at 45 degree positions relative to the position of robot arm 630 .
- the robot arm 630 can place a sample plate 10 at staging position 632 , in preparation for placing the sample plate 10 in load position 202 .
- the robot can move the read plate from load position 202 to staging position 634 , then load the plate from staging position 632 to load position 202 , and while the sample plate 10 is being imaged, the robot can move the plate from staging position 634 to the discard stack 628 , and place a new sample plate 10 at staging position 632 .
- the staging positions are at approximately the same level as the load position, so movement is very quick.
- the robot arm 630 can do the time consuming moves to any of robot plate stacks 620 , 622 , 624 , and 626 while imaging is going on, rather than in series with imaging.
- the cycle time for a single sample plate 10 is 2 moves from/to staging areas (3 seconds each), plus 2 transport moves IN/OUT to read position 203 (3 seconds each), plus the integration time (image exposure) time (typically 60 seconds), for a total cycle time of 72 seconds.
- the time would be 2 moves to stacks (30 seconds each), plus 2 transports (3 seconds each), plus the integration time (typically 60 seconds) for a total of 126 seconds.
- the use of staging positions 632 and 634 decreases cycle time by 43%.
- the mechanical and optical systems of the luminometer workstation of the invention are designed to provide precise, quantified luminescent values in an HTS environment, taking advantage of the use of a Fresnel lens/collimator assembly to permit single image viewing by the CCD camera, and subsequent analysis.
- the collimator, the lens and the camera together combine to reduce cross-talk experienced in prior art attempts.
- the signals obtained are further processed, as illustrated in FIG. 7 , through software loaded onto processor 50 , or other convenient method, to further refine the values obtained.
- the integrated processing component of the invention Prior to processing image data collected through the integrated mechanical and optical systems of the invention herein described, the integrated processing component of the invention must first control the mechanical alignment of those integrated mechanical and optical systems for reliable data collection. This process is conducted under control of the processor 50 .
- the luminescence detection of the present invention measures the light emitted from four test sample wells, called hot wells, of a test plate.
- the hot wells are located near each corner of the sample tray used for the alignment testing.
- the adjacent well crosstalk from each of the four hot wells is analyzed, and the values are compared. When the collimator is aligned precisely over the sample well tray, the crosstalk values will be symmetrical for the four hot wells.
- the software of the present invention flags any errors detected, such as incorrect number of test sample wells, incorrect intensity, or incorrect location.
- the software of the present invention performs a symmetry calculation to determine precise alignment of the sample well tray, collimator, Fresnel lens and CCD camera assembly.
- known software techniques are employed to perform the symmetry calculation process by performing the following steps:
- step A three actual images for each filter/emitter are taken.
- a 1 is a precursor image
- a 2 is the full integration time image
- a 3 is post-cursor image.
- the precursor and post-cursor images are taken to avoid the problem of pixel saturation and to extend the detection dynamic range.
- the precursor and post-cursor images refer to reduced integration time images, which should not contain multiple saturated pixels. If more than six pixels of the full integration time image are saturated, the pre- and post-cursor images are averaged together to form the actual data for that well area. In the absence of six pixel saturation, the full integration time image is used.
- each image is subjected to edge detection and masking, a processing step whereby the edge of each well or corresponding light image is identified, or annotated, to set off and clearly separate each well region of interest, as disclosed in U.S. patent application Ser. No. 09/351,660, incorporated herein by reference.
- edge detection and masking is performed for each of B 1 , B 2 and B 3 , referring to the pre-cursor, full integration time image and post-cursor images, respectively.
- the images are then subjected to “outlier” correction, correcting or “shaving” outliers and anomalies.
- the pixels within the region of interest are examined to identify “outliers”—those that are in gross disagreement with their neighbors, in terms of light intensity detected, and if the intensity of a given pixel or small pixel area is significantly different than neighboring pixels or pixel areas, then the average of the surrounding pixels or areas is used to replace erroneous data. This can be due to random radiation, such as that caused by cosmic rays. In this process, this type of intensity is corrected.
- each image C 1 , C 2 and C 3 is subjected to dark subtraction, subtracting the dark background, so as to obtain average pixel values within each mask-defined region of interest.
- the subtraction is done on a well-by-well basis from stored libraries which are updated periodically.
- the dark subtraction is conducted to correct for the fact that even in the absence of light, CCD cameras can output low level pixel or bin values.
- This value includes the electronic bias voltage, which is invariant of position and integration time, and the “dark current,” which may vary by position, and is proportional to integration time and to the temperature of the CCD.
- the CCD may also have faulty pixels that are always high level or saturated regardless of light input.
- the processing software of the invention subtracts this background image or data from the real sample well image data in step C.
- it is known to take a “dark” image immediately before or after a real image, imaging for the same integration time in both cases, and subtracting the “dark” image data from the real image data.
- “dark” image data is collected intermittently, preferably at specific time intervals.
- the initial “dark” image background data is collected at startup, and then typically at four hour intervals during image processing operations.
- the background image has an integration time-invariant component and an integration time-variant component
- data is collected for each sample well at minimum integration time and at maximum integration time, and a “slope/intercept” line is calculated between the two data points, using known data analysis techniques. This calculation permits data interpolation for any integration time between the minimum and maximum, and also permits data extrapolation for integration times below or beyond the minimum and maximum integration times.
- a CCD camera is employed that has two separate “dark” current functions, caused by the CCD output amplifier. Operation of the amplifier generates heat and necessarily creates background “dark” image data. In the preferred embodiment, for integration times of less than 10 seconds, the amplifier operates continuously, whereas for integration times of more than 10 seconds, the amplifier remains off until immediately prior to the read operation. The “slope/intercept” line calculated for integration times of more than 10 seconds will then necessarily have a lower slope than a “slope/intercept” line calculated for integration times of less than 10 seconds.
- the processing software element allows separate collection and least squares regression for both the 0 to 10 second integration time region a processor 50 , the “dark” background image data is stored separately for each individual AOI.
- “Dark” current and bias can also vary over time.
- the processing software element corrects for this effect by comparing the integration time normalized (using the regression line technique described above) “dark reference” pixel values (outside the imaging field-described above), that were taken when the “dark” background images were taken, versus the “dark reference” pixel values taken while real sample well images are being taken. The difference between the values is then subtracted or added, as applicable, as a global number, to the “dark” background data. This corrects for bias drift and also for global CCD temperature drift.
- step C all of the above “dark background” interpolation/subtraction of step C is done on a well by well basis.
- step D if pixel saturation has occurred such that the average of the pre-cursor and post-cursor image must be used, the image data is multiplied by the reciprocal of the percentage represented by the pre-cursor images (e.g., 3%).
- step E the well data is corrected for uniformity variations using a calibration file that is the reciprocal of the system response to a perfectly uniform input illumination.
- step F the cross-talk correction is effected by processing the data as a whole and preparing a final image in much the same fashion as reconstruction of three dimensional images from a two dimensional data array is practiced.
- the impulse response function is collected for all 96 wells of the 96 well plate type. This is done by filling one particular well in a given plate with a high intensity luminescent source, imaging the plate, and analyzing all of the wells in the plate for their response to the one high intensity well.
- the IRF is collected for all of the wells individually by repeating the process for every different well location desired for the complete data set. For 384 plate types, 96 sampling areas are selected, and data for the wells in between the selected sampled areas are interpolated in two dimensions. In the preferred embodiment, the 96 sampling areas comprise every second row and every second column, starting at the outside and working toward the center.
- the two center rows and the two center columns are interpolated.
- the reflections in a 384 well plate are also modeled, and used to predict and interpolate reflections for the missing input data. Further in the preferred embodiment, all wells are normalized to the well with the highest intensity.
- step F the two-dimensional array of well IRF values for each welfare “unfolded” into a one-dimensional column array, and the two-dimensional arrays of IRF values for other wells are added as subsequent columns, as shown in Chart 1 following:
- This unfolded matrix is then inverted, using known matrix inversion techniques, and used as a correction to matrix multiply a one-dimensional matrix unfolded from real assay data.
- This arithmetic process may be shown as matrix algebra:
- the calculated well intensities resulting from the above processing are calibrated to an absolute parameter of interest, such as the concentration of a known reporter enzyme.
- an absolute parameter of interest such as the concentration of a known reporter enzyme.
- the processed image information is subjected to any necessary post adjustment processing, for appropriate correlation with the materials tested.
- the processing software of the present invention is capable of performing multi-component analysis.
- the basic problem is to calculate separately the concentration of a single reagent in a single sample containing other different reagents.
- the reagents used with the invention are formulated so as to emit over different, but perhaps overlapping, spectrums.
- the first step of separating the light from multiple reagents is accomplished by optical bandpass filters, which are designed to maximize the sensitivity of the target reagent emission, while minimizing the sensitivity to other non-target reagent emission.
- optical filters are interference devices, their bandpass characteristics vary, dependent on the angle of incidence of the emission to be filtered.
- the angle of incidence will be unique for each well because each well's specific location is unique relative to the optical filter. Accordingly, all calculations and filter coefficients must be unique per sample well.
- the multi-component calibration is performed as follows:
- the target reagent for that filter should produce the highest output.
- the other reagents may also have spectra in the filter's bandpass, and will produce smaller outputs, which are a measure of the overlap of those nontarget reagent spectra into the filter signal.
- the filter's output for the target reagent might be 850
- the filter's output for the other 2 reagents might be 100 and 50, respectively.
- the total output would be 1000, and the proportions would be 850:100:50.
- These coefficients are measured for each well location and filter separately, which gives a complete set of coefficients for simultaneous equations. This will allow a solution for any combination of concentrations of reagent in one sample well. Further in the preferred embodiment, these coefficients will also be normalized by the total intensity read in the “total emission” filter, so that the calculation will result in the same intensity as the instrument would measure if only a single reagent was measured by the “total emission” filter. This calculation may be shown as follows for a simple case of blue and green reagents (abbreviated as R in the calculations), and blue and green and total emission filters (abbreviated as F in the calculations):
- step G the raw output of the instrument for each filter is normalized for integration time before solving the equations.
- step H the analyzed data is presented in a user-acceptable format, again controlled by processor 50 .
- the invention may be further understood by reference to examples of assays practiced in HTS format, demonstrating the dynamic range and flexibility of the NorthStarTM luminometer.
- cAMP standards were serial diluted and added to a 96-well assay plate with alkaline phosphatase conjugated cAMP and anti-cAMP. Plates were processed with the cAMP-ScreenTM protocol and imaged for 1 minute on the NorthStarTM 30 minutes after addition of CSPD®/Sapphire-IITM. A sensitivity of 0.06 pM of purified cAMP is achieved with cAMP-ScreenTM on the NorthStarTM workstation. The results are depicted in FIG. 8 .
- Adrenergic ⁇ 2 Receptor-expressing C2 cells were plated in a 96-well plate (10,000 cells/well) and stimulated with isoproterenol for 10 minutes. cAMP production was quantitated in cell lysates using the cAMP-ScreenTM assay. The assay plate was imaged for 1 minute on the NorthStarTM, 30 minutes after addition of CSPD®/Sapphire-IITM. Increasing cAMP levels were detected on the NorthStarTM from the stimulated adrenergic receptor. The results are depicted in FIG. 9 .
- PCRE-Luc contains the luciferase reporter gene under the control of a cAMP response element (CRE).
- CRE cAMP response element
- Forskolin induces intracellular cAMP production through the irreversible activation of adenylate cyclase. All plate formats demonstrate comparable forskolin-induced cAMP levels. The results are depicted in FIG. 10 .
- pCRE-Luc-Transfected cells were seeded in a 96-well plate. Four random wells were induced for 17 hours with 1 mM forskolin and the entire plate was assayed with the Luc-ScreenTM system. The results are shown in FIG. 11 .
- NIH/3T3 cells were co-transfected with pCRE-Luc and p ⁇ gal-Control, and seeded into a 96-well microplate (2 ⁇ 10 4 cells/well). Cells were incubated with forskolin for 17 hours. Modified Dual-Light® Buffer A was added to cells and incubated for 10 minutes. Modified Dual Light® Buffer B was injected and luciferase-catalyzed light emission was measured immediately. Thirty minutes later, Accelerator-II was added, and then ⁇ -galactosidase-catalyzed light emission was quantitated on the NorthStarTM HTS workstation. Quantitation is shown graphically in FIG. 12 .
- CHO-Aeq-5HT2B cells were loaded with coelenterazine h+/ ⁇ 0.5 ⁇ M BAPTA-AM for 4 hours.
- the antagonist methysergide was added to the charged cells for 30 minutes. 1 ⁇ M agonist a-Me-5HT was injected, and the emitted light was integrated for 20 seconds on the NorthStarTM system.
- the reported IC50 for methysergide (0.6 nM) is unchanged in the presence of BAPTA-AM. The data obtained appears in FIG. 14 .
- CHO-Aeq-OX2-A2 cells (Euroscreen) were loaded with coelenterazine h+/ ⁇ 0.6 ⁇ M BAPTA-AM for 4 hours.
- the peptide agonist Orexin B was injected into the wells, and the emitted light was integrated for 20 seconds on the NorthStarTM.
- the reported EC50 for Orexin B (0.75 nM) is unchanged in the presence of BAPTA-AM. This is shown in FIG. 15 .
Abstract
A luminescence detecting apparatus and method for analyzing luminescent samples is disclosed. Luminescent samples are placed in a plurality of sample wells in a tray, and the tray is placed in a visible-light impervious chamber containing a charge coupled device camera. The samples may be injected in the wells, and the samples may be injected with buffers and reagents, by an injector. In the chamber, light from the luminescent samples pass through a collimator, a Fresnel field lens, a filter, and a camera lens, whereupon a focused image is created by the optics on the charge-coupled device (CCD) camera. The use of a Fresnel field lens, in combination with a collimator and filter, reduces crosstalk between samples below the level attainable by the prior art. Preferred embodiments of the luminescence detecting apparatus and method disclosed include central processing control of all operations, multiple wavelength filter wheel, and robot handling of samples and reagents. Preferred embodiments of processing software integrated with the invention include elements for mechanical alignment, outlier shaving, edge detection and masking, manipulation of multiple integration times to expand the dynamic range, crosstalk correction, dark subtraction interpolation and drift correction, multi-component analysis applications specifically tailored for luminescence, and uniformity correction.
Description
- This application claims the benefit from Provisional Application Ser. No. 60/144,891, filed Jul. 21, 1999. The entirety of that provisional application is incorporated herein by reference.
- 1. Field of the Invention
- This invention relates to the field of apparatus and methods for detecting and quantifying light emissions, and more particularly, to detecting and quantifying light emitted from luminescent-based assays. Still more particularly, this invention pertains to apparatus and methods for detecting and quantifying luminescence such as bioluminescence and/or chemiluminescence from luminescent assays as an indicator of the presence or amount of a target compound. Preferred embodiments of the invention include as an imaging device a charge coupled device (CCD) camera and a computer for analyzing data collected by the imaging device. Further preferred embodiments include the capacity for use in high throughput screening (HTS) applications, and provide for robot handling of assay plates.
- 2. Description of the Related Art
- The analysis of the luminescence of a substance, and specifically the analysis of either bioluminescence (BL) or chemiluminescence (CL), is becoming an increasingly useful method of making quantitative determinations of a variety of luminescent analytes.
- Recently, methods have been introduced that utilize luminescence detection for quantitatively analyzing analytes in an immunoassay protocol. Such luminescence immunoassays (LIA) offer the potential of combining the reaction specificity of immunospecific antibodies or hybridizing nucleic acid sequences and similar specific ligands with the high sensitivity available through light detection. Traditionally, radioactive reagents have been used for such purposes, and the specificity and sensitivity of LIA reagents is generally comparable to those employing traditional radiolabelling. However, LIA is the preferred analytical method for many applications, owing to the nontoxic nature of LIA reagents and the longer shelf lives of LIA reagents relative to radioactive reagents.
- Among other luminescent reagents, chemiluminescent compounds such as 1,2-dioxetanes, developed by Tropix, Inc. and other stable chemiluminescent molecules, such as xanthan esters and the like, are in commercial use. These compounds are triggered to release light through decomposition triggered by an agent, frequently an enzyme such as alkaline phosphatase, which is present only in the presence, or specific absence, of the target compound. The detection of light emission is a qualitative indication, and the amount of light emitted can be quantified as an indicator of the amount of triggering agent, and therefore target compound, present. Other well known luminescent compounds can be used as well.
- Luminescent release may sometimes be enhanced by the presence of an enhancement agent that amplifies or increases the amount of light released. This can be achieved by using agents which sequester the luminescent reagents in a microenvironment which reduces suppression of light emission. Much biological work is done, perforce, in aqueous media. Water typically suppresses light emission. By providing compounds, such as water soluble polymeric onium salts (ammonium, phosphonium, sulfonium, etc.) small regions where water is excluded that may sequester the light emitting compound may be provided.
- The majority of instrumentation used to monitor light emitting reactions (luminometers) use one or more photomultiplier tubes (PMTs) to detect the photons emitted. These are designed to detect light at the low light levels associated with luminescent reactions. The rate at which a PMT based microplate luminometer can measure signal from all wells of the plate is limited by the number of PMTs used. Most microplate luminometers have only one PMT so a 384 well plate requires four times longer than is required to read a 96-well plate.
- The nature of biological research dictates that numerous samples be assayed concurrently, e.g., for reaction of a chemiluminescent substrate with an enzyme. This is particularly true in gene screening and drug discovery, where thousands of samples varying by concentration, composition, media, etc. must be tested. This requires that multiple samples be reacted simultaneously, and screened for luminescence. However, there is a need for high speed processing, as the chemiluminescence or bioluminescence may diminish with time. Simultaneously screening multiple samples results in improved data collection times, which subsequently permits faster data analysis, and contingent improved reliability of the analyzed data.
- In order for each specific sample analyte's luminescence to be analyzed with the desired degree of accuracy, the light emission from each sample must be isolated from the samples being analyzed concurrently. In such circumstances, stray light from external light sources or adjacent samples, even when those light levels are low, can be problematic. Conventional assays, particularly those employing high throughput screening (HTS) use microplates, plastic trays provided with multiple wells, as separate reaction chambers to accommodate the many samples to be tested. Plates currently in use include 96- and 384-well plates. In response to the increasing demand for HTS speed and miniaturization, plates having 1,536 wells are being introduced. An especially difficult impediment to accurate luminescence analysis is the inadvertent detection of light in sample wells adjacent to wells with high signal intensity. This phenomenon of light measurement interference by adjacent samples is termed ‘crosstalk’ and can lead to assignment of erroneous values to samples in the adjacent wells if the signal in those wells is actually weak.
- Some previously proposed luminometers include those described in U.S. Pat. No. 4,772,453; U.S. Pat. No. 4,366,118; and European Patent No. EP 0025350. U.S. Pat. No. 4,772,453 describes a luminometer having a fixed photodetector positioned above a platform carrying a plurality of sample cells. Each cell is positioned in turn under an aperture through which light from the sample is directed to the photodetector. U.S. Pat. No. 4,366,118 describes a luminometer in which light emitted from a linear array of samples is detected laterally instead of above the sample. Finally, EP 0025350 describes a luminometer in which light emitted through the bottom of a sample well is detected by a movable photodetector array positioned underneath the wells.
- Further refinements of luminometers have been proposed in which a liquid injection system for initiating the luminescence reaction just prior to detection is employed, as disclosed in EP 0025350. Also, a temperature control mechanism has been proposed for use in a luminometer in U.S. Pat. No. 4,099,920. Control of the temperature of luminescent samples may be important, for example, when it is desired to incubate the samples at an elevated temperature.
- A variety of light detection systems for HTS applications are available in the market. These include the LEADseeker™ from Amersham/Pharmacia, the ViewLux™ offered by PerkinElmer and CLIPR™ from Molecular Devices. These devices are all expensive, large dimensioned (floorbased models), exhibit only limited compatibility with robotic devices for plate preparation and loading, have a limited dynamic range, and/or use optical detection methods which do not reduce, or account for, crosstalk. The optical systems used are typically complex teleconcentric glass lens systems, which may provide a distorted view of wells at the edges of the plates, and the systems are frequently expensive, costing in excess of $200,000.00. Perhaps the most popular detection apparatus is the TopCount™, a PMT-based detection system from Packard. Although the TopCount™ device has a desirable dynamic range, it is not capable of reading 1,536 well plates, and it does not image the whole plate simultaneously.
- Crosstalk from adjacent samples remains a significant obstacle to the development of improved luminescence analysis in imaging-based systems. This can be appreciated as a phenomenon of simple optics, where luminescent samples produce stray light which can interfere with the light from adjacent samples. Furthermore, the development of luminometers capable of detecting and analyzing samples with extremely low light levels are particularly vulnerable to crosstalk interference.
- In order to meet the above-identified needs that are unsatisfied by the prior art, it is a principal object and purpose of the present invention to provide a luminescence detecting apparatus that will permit the analysis of luminescent samples. It is a further object of the present invention to provide a luminescence detecting apparatus capable of simultaneously analyzing a large number of luminescent samples. In a preferred embodiment of the present invention, a luminescence detecting apparatus is provided that simultaneously analyzes multiple samples held in wells, where the well plates contain as many as 1,536 wells. The present invention further includes robot handling of the multiple well trays during analysis.
- It is yet another object of the present invention to provide a luminescence detecting apparatus capable of analyzing low light level luminescent samples, while minimizing crosstalk from adjacent samples, including and especially minimizing crosstalk from adjacent samples with higher light level output than the sample to be analyzed.
- The apparatus of this invention employs a Fresnel lens arrangement, with a vertical collimator above the well plate, with dimensions to match the number of wells. Thus, a 1,536-well plate will employ a dark collimator above the plate with 1,536 cells in registry with the wells of the plate. Fixed above the collimator is a Fresnel lens, which refracts the light such that the view above the lens appears to be looking straight down into each well, regardless of its position on the plate, even at the edges.
- Above the Fresnel lens is a CCD camera arranged so as to take the image of the entire plate at one time, viewing through a 35 mm wide angle lens, to give whole plate imaging on a rapid basis. Between the CCD and Fresnel/collimator is a filter, typically arrayed on a filter wheel, disposed at an angle to the lens. The filter is selected to permit the passage of the specific wavelength of the light emitted, and reflect or absorb all others. Several filters may be provided on the wheel, to permit sequential detection of light emitted from multiple reagents emitting light at different wavelengths.
- The samples are fed to the optical detection platform through a loading device designed to work well with robotic and automated preparation systems. The well-plate, with reaction mixture already provided, is placed on a shuttle by a human, or preferably, robot. Alignment of the plate on the shuttle may be relatively coarse, notwithstanding the requirement for tight tolerances to match the collimator grid array. As the shuttle leaves the loading position, a resilient means urges the plate into strict conformal alignment. The shuttle positions the plate under an overhead injection bar, which may accommodate up to sixteen wells in a column at one time. If not previously added, a triggering agent or luminescent reagent is added to the sample wells, and the plate indexes forward to load the next column of wells across the plate. The shuttle then advances through a door into the sample chamber, and the plate is aligned with the collimator and the Fresnel lens. Since many reactions proceed better, or only, at elevated temperatures, the sample chamber is insulated, and provided with heating means, for heating the air in or provided to the chamber. In order to maintain temperature in the chamber close to room temperature and to accurately control temperature, the chamber may also be provided with a heat exchanger.
- The light emission from the entire multiple well plate is imaged at once, with subsequent imaging through a different filter if multiple wavelengths are employed. The signal obtained is processed to further reduce crosstalk reduced by the collimator and the presence and amount of luminescence is quickly detected and calculated by a personal computer using automated software. Data is then reported as intensity per well or further analyzed relative to specific assay standards.
- A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
-
FIG. 1 is a cross section of a preferred embodiment of a luminescence detecting apparatus according to the present invention; -
FIG. 2 is a detailed cross section of the optics of a luminescence detecting apparatus according to the present invention. -
FIG. 3 is a cross-sectional view of the plate transport system of the invention. -
FIG. 4 is a perspective illustration of the injector arm assembly of the invention. -
FIG. 5 is an exploded view of the filter wheel assembly. -
FIG. 6 is a cross-sectional view of the optical housing. -
FIG. 6A is a plan view of a robotic mechanism of the invention. -
FIG. 7 is a flow chart illustration of the processing method of the invention. -
FIGS. 8-15 are illustrations of the results obtained using the invention in Examples 1-10, respectively. - Referring now to the drawings, wherein like reference numerals designate identical or corresponding parts throughout the several views, and more particularly to
FIG. 1 thereof, a preferred embodiment of the luminescence detecting apparatus of the present invention uses a shuttle or tray to carry a micro plate (plate) 10 comprising a plurality ofsample wells 20 which may in the preferred embodiment number as many as 1,536 or more. Persons of ordinary skill in the relevant art will recognize that the number ofsample wells 20 is limited only by the physical dimensions and optical characteristics of the luminometer elements, and not by the technology of the present invention. Thesample wells 20 may be filled with analyte manually, or robotically prior to delivery to the inventive apparatus. Agents necessary for chemiluminescence may be filled automatically via theinjector 30, to which analyte is supplied through an array ofsupply tubes 40 or prior to placing the plate on the tray. Typically, the sample wells will contain chemiluminescent reagents. These reagents emit light at intensities proportional to the concentration of analyte in the sample. This light can be very low intensity and requires an instrument with sufficient sensitivity to achieve the desired detection limits. - The operation of
injector 30 is controlled bycentral processor 50, which in the preferred embodiment may control the operation of all elements of the luminometer of the present invention. Data collection, analysis and presentation may also be controlled byprocessor 50. Further in a preferred embodiment of the present invention, theinjector 30 may also be used to add buffer solutions to the analytes and also to add reagents that enable “glow” and/or “flash” luminescence imaging, that is sustained or brief, intense emission, respectively, all under control ofcentral processor 50. - After the analytes are placed in the
sample wells 20,plate 10 is placed insample chamber 55, which is located inoptical chamber 60 at a fixed focal distance from and directly under the charge-coupled device (CCD)camera 70, in order to permit the CCD camera to image the luminescent sample accurately. Thesample chamber 55 is preferably capable of precise temperature control, as many luminescent reagents and specific luminescent reactions are temperature dependent. Temperature control is provided bycentral processor 50, which can vary the temperature for eachindividual sample plate 10, ascentral processor 50 controls the movement and injection of thesample wells 20 in eachsample tray 10. In a preferred embodiment of the luminometer of the present invention,central processor 50 also controls an industrial robot (not shown) which performs the activities involving analyte handling in the luminometer of the present invention. - With the
plate 10 placed in thesample chamber 55, theoptics 80 deliver the image of thecomplete microplate 10 as a single image to theCCD camera 70. - Although the operation of the luminometer of this invention is an integral, continuous practice, and all elements of the luminometer cooperate together to provide precise, accurate and reliable data, the invention may be more easily understood by reference to three separate, integrated systems, the optics system, the mechanical system and the processing system. Each is discussed in turn, with a discussion of examples of the operation as a whole to follow.
- Turning now to
FIG. 2 , theoptics 80 are shown in further detail.Luminescent emission 100 from the analyte in plate well 20 located in theplate 10 travels first throughdark collimator 110, which permits only parallel and semi-parallel light rays to exit thesample wells 20 for eventual imaging by theCCD camera 70. The effect of collimation assists with the prevention of stray light from thesample wells 20 and with the elimination of crosstalk between luminescent samples. Thecollimator 110 may be sealably engaged, or in close proximity, to thesample tray 10, to enhance the restriction of stray light from the samples. Each well 10 is in strict registration and alignment with a corresponding grid opening incollimator 110. From thecollimator 110, the luminescent radiation passes through aFresnel field lens 120, which focuses the light towardfilter 130. In a preferred embodiment of the present invention, thecollimator 110 andFresnel field lens 120 are packaged in a cassette that can be changed by the user. Such an equipment change may be necessitated by varying optical characteristics of different analytes and different well distributions in plates. - The use of a Fresnel field lens is preferable to alternative optical devices for several reasons. Initially, improvements in design and materials have capitalized on the superior optical capabilities of the Fresnel lens, while virtually eliminating its once inherent limitations. Today, many Fresnel lenses are made of molded plastic, creating an almost flawless surface with very little scatter light. The elimination of scatter light is an important element of eliminating crosstalk between adjacent samples in the luminometer of the present invention. Furthermore, improved types of plastics commonly employed in the manufacturing of Fresnel lenses and other optical devices have optical qualities equivalent to ground glass lenses.
- Using high tech processes such as computer-controlled diamond turning, complex aspheric surfaces can be cut into a long lasting mold for casting Fresnel lenses. In this manner, Fresnel lenses can be manufactured to produce the precise optical imaging effect that is most efficient for a charge coupled device camera, as in the present invention. Also, Fresnel lenses offer an advantage over conventional lenses in that they can be molded flat and very thin. Because of the shape of the Fresnel lens, it can easily be integrated directly into the housing of the luminometer, enhancing the light-tight properties necessary for accurate imaging of low light samples. Furthermore, Fresnel lenses are much less expensive than comparable conventional glass lenses.
- As with any other lens, the total beam spread from a Fresnel lens depends on the size of the source in relation to the focal length of the lens. Smaller sources, such as luminescent assay samples, and longer focal lengths produce more compact beams. Since there are practical limitations to minimizing the geometry and dimensions of the
optics 80 in the luminometer of the present invention, the use ofFresnel field lens 120 provides the greatest opportunity for fine-tuned optics. The emissions fromplate 10 pass throughlens 120, and are refracted such that the image obtained atCCD 70 appears to look directly downward into all wells, even laterally displaced (edge) ones. This feature is typically called “telecentric.” - Further in a preferred embodiment of the present invention, the
filter 130 may be configured on a wheel, wherein different filter elements may occupy different portions of the wheel, depending on the luminescent characteristics of the sample being analyzed.Filter 130 is preferably inclined at an angle of 20°-30° relative to the CCD, so that stray reflected light is reflected outside the field of view. Specifically, thefilter wheel 130 permits the selection of different wavelength ranges, which not only permit high quality imaging, but may be used to separate the emissions of different reagents emitting at different wavelengths. Again, thefilter wheel 130 is controlled bycentral processor 50, in coordination withcentral processor 50's control of theindividual sample wells 20 in thesample plate 10. In many assays, such as those addressed in pending U.S. patent application Ser. No. 08/579,787, incorporated by reference herein, multiple luminescent reagents, which emit at different wavelengths, are employed in a single well. Using multiple filters, each can be imaged in turn, and the true concentration can be calculated from the data set resulting using pre-stored calibration factors.Filter 130 is preferably provided with an infrared (IR) filter operating in conjunction with the selected bandpass, or as an independent element. Applicants have discovered that stray IR radiation, resulting from the plate phosphorescence, resulting in abnormally high backgrounds. An IR filter suppresses this. - From the
filter wheel 130, the sample-emitted light passes throughcamera lens 140, which in the preferred embodiment is a large aperture, low distortion, camera lens.Camera lens 140 focuses the image of the sample on theCCD chip 70. In the preferred embodiment of the present invention,CCD camera 70 is a cooled, low noise, high resolution device. The lens is preferably a 35 mm wide angle lens with a low light level (F1.4) large aperture character. Magnification of 3-6, preferably about 5.5, is preferred. In preferredembodiments CCD camera 70 is provided with an anti-blooming CCD chip, to enhance dynamic range, which is about 105 in the claimed invention, referred to as the NorthStar™ luminometer. Blooming occurs when a single pixel is overloaded with light and its photoelectrons overflow the CCD device well capacity, obliterating surrounding pixels. Further in the preferred embodiment of the luminometer of the present invention, the selected CCD camera includes a liquid cooled thermoelectric (Peltier) device providing cooling of the CCD to approximately −35° C., and the CCD has 1280×1024 pixels, each of which are 16 μm square, producing a total active area of 20.5 mm×16.4 mm. The quantum efficiency averages 15% over the range from 450 nanometers to 800 nm. The output is digitized to 16 bit precision and pixels can be “binned” to reduce electronic noise. - By using the features disclosed herein, the luminometer of the present invention has a spatial resolution capable of providing high quality imaging of high density, sample trays. The noise performance and CCD temperature are designed to provide the desired detection limit.
- The mechanical systems of the luminometer workstation of this invention are designed to achieve automated, high throughput precise delivery of microplates in registration with a
collimator 110 so as to be read by theCCD Camera 70. To this end, as shown inFIG. 3 , a cross-section of theinventive luminometer shuttle 200 translates from aload position 202, whereplates 10 are loaded on to the shuttle, preferably by a robotic device such as robot arm, and theshuttle 200 then translates towardssample chamber 55, to readposition 203.Shuttle 200 is caused to translate by a conventional stepper motor (not pictured). Asshuttle 200 advances towardsample chamber 55, it may stop underneathinjector 30.Injector 30 is more fully illustrated below inFIG. 4 . Referring still toFIG. 3 ,injector 30 delivers fluid reagents drawn fromreservoir 204.Syringe pump 205 draws the fluid reagents fromreservoir 204, and pumps the fluid to theinjector tubes 40. Twoway valve 206 controls the passage of the fluid drawn bysyringe pump 205 fromreservoir 204 and pumped bysyringe pump 205 to thesupply tubes 40. In actual practice, there are asmany injector tubes 40 as injection ports being used, and multiple syringe pumps 205 are also used. As will be shown below inFIG. 4 ,injector 30 has up to sixteeninjection ports 302. The plates used in conjunction with the luminometer when injection is used are typically prepared with up to sixteen wells in a column. As theshuttle 200 advancesplate 10 underneathinjector 30,shuttle 200 stops so that thefirst column 208 of wells is directly aligned underinjector 30. Precise amounts of analyte are delivered to the first set of wells, andshuttle 200 indexes forward one column, so as to inject reagent into the second column of wells 210. This process is repeated until all wells are filled. Thereafter,shuttle 200 advances forward intosample chamber 55 through hinged door 212. In the alternative, door 212 may be a guillotine door or similar type of closing mechanism. The wells ofplate 10 are then read insample chamber 55. Upon completion of reading,shuttle 200 translates back toload position 202. - Before
shuttle 200 advances to the injection bar, it may be necessary to fully prime the tube with fluid, so as to provide for precise delivery into the plate.Trough 304 swings out from its storage position parallel to the direction of travel ofshuttle 200, shown by an arrow, to a position directly underlying theinjector 30, perpendicular to the direction of travel. Fluid in the injector andtubes 204 are delivered intotrough 304, and removed by suction.Trough 304 then returns to its rest position, parallel to, and away from, the direction of travel of theshuttle 200, when the shuttle is moved toward thesample chamber 55. On its return trip to loadposition 202,locator 214 onshuttle 200 is engaged bycam 216.Locator 214 is mounted on a resilient means, such that when engaged bycam 216, thelocator 214 recesses away fromplate 10. This permits removal ofplate 10, and delivery from a robotic arm or other source of afresh plate 10, without the requirement of precise location. Asshuttle 200 moves away fromload 202,locator 214 is urged forward, firmly locatingplate 10 in place.Plate 10 is held againstshoulder 217 by the resilient urging oflocator 214. - It is important that each plate be precisely identified, so that results are correlated with the correct test samples. In most HTS laboratories, most microplates are labeled with a unique “bar code.” The label is often placed on the surface perpendicular to the plane of the plate itself. To permit precise identification of each plate, a
bar code reader 218 is mounted on the luminometer housing generally indicated at 299 and directly above the door 212, for example on an arm orflange 220.Bar code reader 218 is focused on amirror 222 which in turn permits reading directly off the front or leading edge ofplate 10 as it approaches onshuttle 200. Thus, before each plate arrives in the sample chamber, its identity has been precisely recorded inprocessor 50, and the results obtained can be correlated therewith. Persons of ordinary skill in the art will recognize that a variety of configurations of alignment and placement of bothbar code reader 218 andmirror 222 will result in the desired identification. - As more clearly shown in
FIG. 4 ,injector 30 may be precisely located by operation ofactuator wheel 306, provided with positions corresponding to the total number of wells on the plates being assayed. Similarly, the vertical position, to account for the different thicknesses of the plate, may be controlled bywheel 308. Given the simple translation movement ofshuttle 200, and the precise locating and identification of each plate carried, rapid cycling of micro-plate test plates into and out ofsample chamber 55 can be effected. - As described above in connection with the optics system of the invention, a filter is provided which includes or reflects passage of light other than light falling within the selected wavelength of the luminescent emitter in use. The filter assembly is illustrated in exploded format in
FIG. 5 .Filter frame 502 is supported byarm 504 which is connected to the hub of thefilter wheel 506. Multiple different filters may be provided on a single wheel. The filter itself, 508, is securely mounted on the frame and held there bycover 510, which is secured to frame 502 by grommets, screws or other holdingdevices 512. As noted, filter wheel is positioned so as to holdfilter 508 inframe 502 at in incline with respect tocollimator 110, of about 22° nominally, so as to direct any reflections outside the field of view. Light passes through thefilter opening 514, in alignment withcamera lens 140 andCCD camera 70. As further noted above, filter 508 preferably includes an infrared block, either as a component of the filter itself, or as a component provided in addition to the filter for the measured light. An IR block is of value to prevent infrared emissions caused by extraneous radiation from altering the image received by the CCD camera. -
Optical chamber 60 is more fully illustrated inFIG. 6 . As shown,optical chamber 60 is bounded byoptical housing 602 in which fitssample housing 604. When aplate 10 is loaded intooptical chamber 60, the plate is secured insample housing 604 which is positioned in registry withcollimator 110, over which is providedFresnel lens 120. While many luminescent assays can be provided at ambient temperatures, some require elevated temperatures. The luminometer of this device is provided with a sample chamber in which thesample housing 604 carriesinsulation 606 which, in a preferred embodiment is polyurethane foam, andheater element 608 to raise the temperature in thesample chamber 55 above ambient temperature, up to about 42° C. - There is a tendency, even at ambient conditions, for condensation to collect on the surface of the
Fresnel lens 120, as a result of moisture coming from the filled wells ofplate 10. Thedefogger 610 directs a stream of air heated just a few degrees, preferably about 2-3° degrees, above ambient conditions, or above the temperature of the chamber if the chamber is above ambient conditions, across the surface of theFresnel lens 120, effectively preventing condensation. Mounted at the top of the interior ofoptical chamber 60 isfilter motor 610 which drivesfilter wheel 612, on which may be mountedfilters 614 of varying wavelength, for filtering undesirable wavelengths prior to imaging. Of course, a region is provided, indicated at 616, in theoptical housing 602 of theoptical chamber 60 for light to be directed onto the CCD camera after passing through thefilter 614. The dimensions ofoptical chamber 60 are exaggerated inFIG. 6 to illustrate the relationship between theoptical chamber 60 and thefilter wheel 612, anddefogger 610. In practice, the filter is located inside theoptical chamber 60, and outside thesample housing 604 but alternate locations are possible while still achieving the desired function. - In
FIG. 6A , a plan view of a novelrobotic mechanism 616 is displayed in a preferred embodiment of the present invention, which provides capacity for use in high throughput screening (HTS) applications. Referring toFIG. 6A , the operation is as follows: robot plate stacks 620, 622, 624, 626, and 628 each can be filled withmultiple sample plates 10, arranged in a vertical stack. In the preferred embodiment ofFIG. 6A ,robot plate stack 628 is designated as the discard stack. The remaining robot plate stacks 620, 622, 624, and 626 can be programmed in order of delivery by software controlled by processor 50 (not shown). In order to load or pick plates from any of these stacks,robot arm 630 moves vertically and rotationally to the desired robot plate stack, under control of the software programmed inprocessor 50. - When commanded by
processor 50,transport 200 of the instrument will move thesample plate 10 fromload position 202 to theRead position 203, and return it to loadposition 202 when imaging is complete. In the embodiment of the invention shown inFIG. 6A , the elapsed time between moving thesample plate 10 fromload position 202 to theread position 203, and returning it to loadposition 202 is typically 30-120 seconds, including imaging time. - Staging positions 632 and 634 are located at 45 degree positions relative to the position of
robot arm 630. In one embodiment, while imaging is in process, therobot arm 630 can place asample plate 10 at stagingposition 632, in preparation for placing thesample plate 10 inload position 202. When the imaging is complete, the robot can move the read plate fromload position 202 to stagingposition 634, then load the plate from stagingposition 632 to loadposition 202, and while thesample plate 10 is being imaged, the robot can move the plate from stagingposition 634 to the discardstack 628, and place anew sample plate 10 at stagingposition 632. In practice, the staging positions are at approximately the same level as the load position, so movement is very quick. In the preferred embodiment, therobot arm 630 can do the time consuming moves to any of robot plate stacks 620, 622, 624, and 626 while imaging is going on, rather than in series with imaging. - With the staging positions 632 and 634, the cycle time for a
single sample plate 10 is 2 moves from/to staging areas (3 seconds each), plus 2 transport moves IN/OUT to read position 203 (3 seconds each), plus the integration time (image exposure) time (typically 60 seconds), for a total cycle time of 72 seconds. Without usingstaging positions robotic mechanism 616, the use of stagingpositions - As set forth above, the mechanical and optical systems of the luminometer workstation of the invention are designed to provide precise, quantified luminescent values in an HTS environment, taking advantage of the use of a Fresnel lens/collimator assembly to permit single image viewing by the CCD camera, and subsequent analysis. The collimator, the lens and the camera together combine to reduce cross-talk experienced in prior art attempts. The signals obtained are further processed, as illustrated in
FIG. 7 , through software loaded ontoprocessor 50, or other convenient method, to further refine the values obtained. - Prior to processing image data collected through the integrated mechanical and optical systems of the invention herein described, the integrated processing component of the invention must first control the mechanical alignment of those integrated mechanical and optical systems for reliable data collection. This process is conducted under control of the
processor 50. To conduct an alignment test, the luminescence detection of the present invention measures the light emitted from four test sample wells, called hot wells, of a test plate. In a preferred embodiment, the hot wells are located near each corner of the sample tray used for the alignment testing. The adjacent well crosstalk from each of the four hot wells is analyzed, and the values are compared. When the collimator is aligned precisely over the sample well tray, the crosstalk values will be symmetrical for the four hot wells. The software of the present invention flags any errors detected, such as incorrect number of test sample wells, incorrect intensity, or incorrect location. After the detection of no errors or after the correction of detected and flagged errors, the software of the present invention performs a symmetry calculation to determine precise alignment of the sample well tray, collimator, Fresnel lens and CCD camera assembly. In a known embodiment of the invention, known software techniques are employed to perform the symmetry calculation process by performing the following steps: - 1. Extract the hot well and vertical and horizontal adjacent well intensities;
- 2. Calculate the averages of the horizontal and vertical adjacent well intensities separately for each hot well;
- 3. Calculate the differences between the actual adjacent intensity vs. the average for each of the horizontal and vertical directions;
- 4. Normalize the differences by the hot well intensity to convert to a percentage intensity value;
- 5. Find the worst case absolute value of the differences and display that as the overall misalignment;
- 6. Calculate the average X-direction (horizontal) misalignment by averaging the four adjacent wells to the right (horizontal direction) of the hot wells;
- 7. Calculate the average Y-direction (vertical) misalignment by averaging the four adjacent wells to the top (vertical direction) of the hot wells;
- 8. Calculate the rotational misalignment by averaging the left side hot well vertical adjacent wells at the top of the hot wells, and subtracting that from the average of the right side hot well vertical adjacent wells, thereby indicating any tilt in adjacent well values.
- In step A, three actual images for each filter/emitter are taken. A1 is a precursor image, A2 is the full integration time image, and A3 is post-cursor image. The precursor and post-cursor images are taken to avoid the problem of pixel saturation and to extend the detection dynamic range. The precursor and post-cursor images refer to reduced integration time images, which should not contain multiple saturated pixels. If more than six pixels of the full integration time image are saturated, the pre- and post-cursor images are averaged together to form the actual data for that well area. In the absence of six pixel saturation, the full integration time image is used.
- In order to clearly isolate and read each pixel, in step B, each image is subjected to edge detection and masking, a processing step whereby the edge of each well or corresponding light image is identified, or annotated, to set off and clearly separate each well region of interest, as disclosed in U.S. patent application Ser. No. 09/351,660, incorporated herein by reference. Again, edge detection and masking is performed for each of B1, B2 and B3, referring to the pre-cursor, full integration time image and post-cursor images, respectively. The images are then subjected to “outlier” correction, correcting or “shaving” outliers and anomalies. In this process, the pixels within the region of interest are examined to identify “outliers”—those that are in gross disagreement with their neighbors, in terms of light intensity detected, and if the intensity of a given pixel or small pixel area is significantly different than neighboring pixels or pixel areas, then the average of the surrounding pixels or areas is used to replace erroneous data. This can be due to random radiation, such as that caused by cosmic rays. In this process, this type of intensity is corrected.
- Subsequently, in step C, each image C1, C2 and C3 is subjected to dark subtraction, subtracting the dark background, so as to obtain average pixel values within each mask-defined region of interest. The subtraction is done on a well-by-well basis from stored libraries which are updated periodically.
- Specifically, the dark subtraction is conducted to correct for the fact that even in the absence of light, CCD cameras can output low level pixel or bin values. This value includes the electronic bias voltage, which is invariant of position and integration time, and the “dark current,” which may vary by position, and is proportional to integration time and to the temperature of the CCD. The CCD may also have faulty pixels that are always high level or saturated regardless of light input.
- The processing software of the invention subtracts this background image or data from the real sample well image data in step C. As persons of ordinary skill in the relevant art will recognize, it is known to take a “dark” image immediately before or after a real image, imaging for the same integration time in both cases, and subtracting the “dark” image data from the real image data. In the preferred embodiment of the invention, “dark” image data is collected intermittently, preferably at specific time intervals. The initial “dark” image background data is collected at startup, and then typically at four hour intervals during image processing operations.
- Because the background image has an integration time-invariant component and an integration time-variant component, data is collected for each sample well at minimum integration time and at maximum integration time, and a “slope/intercept” line is calculated between the two data points, using known data analysis techniques. This calculation permits data interpolation for any integration time between the minimum and maximum, and also permits data extrapolation for integration times below or beyond the minimum and maximum integration times.
- In a preferred embodiment of the invention, a CCD camera is employed that has two separate “dark” current functions, caused by the CCD output amplifier. Operation of the amplifier generates heat and necessarily creates background “dark” image data. In the preferred embodiment, for integration times of less than 10 seconds, the amplifier operates continuously, whereas for integration times of more than 10 seconds, the amplifier remains off until immediately prior to the read operation. The “slope/intercept” line calculated for integration times of more than 10 seconds will then necessarily have a lower slope than a “slope/intercept” line calculated for integration times of less than 10 seconds. In step C, the processing software element allows separate collection and least squares regression for both the 0 to 10 second integration time region a
processor 50, the “dark” background image data is stored separately for each individual AOI. - “Dark” current and bias can also vary over time. The processing software element corrects for this effect by comparing the integration time normalized (using the regression line technique described above) “dark reference” pixel values (outside the imaging field-described above), that were taken when the “dark” background images were taken, versus the “dark reference” pixel values taken while real sample well images are being taken. The difference between the values is then subtracted or added, as applicable, as a global number, to the “dark” background data. This corrects for bias drift and also for global CCD temperature drift.
- As mentioned, all of the above “dark background” interpolation/subtraction of step C is done on a well by well basis.
- At step D, if pixel saturation has occurred such that the average of the pre-cursor and post-cursor image must be used, the image data is multiplied by the reciprocal of the percentage represented by the pre-cursor images (e.g., 3%).
- In step E, the well data is corrected for uniformity variations using a calibration file that is the reciprocal of the system response to a perfectly uniform input illumination.
- In step F, the cross-talk correction is effected by processing the data as a whole and preparing a final image in much the same fashion as reconstruction of three dimensional images from a two dimensional data array is practiced.
- Specifically in a preferred embodiment of step F, the impulse response function (IRF) is collected for all 96 wells of the 96 well plate type. This is done by filling one particular well in a given plate with a high intensity luminescent source, imaging the plate, and analyzing all of the wells in the plate for their response to the one high intensity well. The IRF is collected for all of the wells individually by repeating the process for every different well location desired for the complete data set. For 384 plate types, 96 sampling areas are selected, and data for the wells in between the selected sampled areas are interpolated in two dimensions. In the preferred embodiment, the 96 sampling areas comprise every second row and every second column, starting at the outside and working toward the center. Because in the 384 well plates the number of rows and columns is even, the two center rows and the two center columns are interpolated. The reflections in a 384 well plate are also modeled, and used to predict and interpolate reflections for the missing input data. Further in the preferred embodiment, all wells are normalized to the well with the highest intensity.
- Subsequently in step F, the two-dimensional array of well IRF values for each welfare “unfolded” into a one-dimensional column array, and the two-dimensional arrays of IRF values for other wells are added as subsequent columns, as shown in
Chart 1 following: -
CHART 1Unfolded Data Into Column 1IRF for IRF for IRF for A1 B1 C1 A1 A1 A1 Etc B1 B1 B1 C1 C1 C1 D1 D1 D1 E1 E1 E1 F1 F1 F1 G1 G1 G1 H1 H1 H1 A2 A2 A2 B2 B2 B2 C2 C2 C2 Etc Etc Etc - The unfolded matrix, which has the form of an N×N matrix, where N=the number of wells to be corrected, comprises a full characterization of the instrument crosstalk, including reflection factors. This unfolded matrix is then inverted, using known matrix inversion techniques, and used as a correction to matrix multiply a one-dimensional matrix unfolded from real assay data. This arithmetic process may be shown as matrix algebra:
-
[true source distribution]×[system IRF]=[instrument output] solving for [true source distribution] produces -
[true source distribution]={1/[system IRF]}×[instrument output] - Subsequently, the calculated well intensities resulting from the above processing are calibrated to an absolute parameter of interest, such as the concentration of a known reporter enzyme. This calibration is conducted through a normalization process producing any of a variety of calibration curves, which will be familiar to those of ordinary skill in the relevant art.
- In optional step G, the processed image information is subjected to any necessary post adjustment processing, for appropriate correlation with the materials tested. Specifically, in a preferred embodiment, the processing software of the present invention is capable of performing multi-component analysis. The basic problem is to calculate separately the concentration of a single reagent in a single sample containing other different reagents. Typically, the reagents used with the invention are formulated so as to emit over different, but perhaps overlapping, spectrums. As earlier described with respect to the integrated optical element, the first step of separating the light from multiple reagents is accomplished by optical bandpass filters, which are designed to maximize the sensitivity of the target reagent emission, while minimizing the sensitivity to other non-target reagent emission. In the present embodiment of the invention, there is one optical filter for each target reagent emission spectrum.
- Since optical filters are interference devices, their bandpass characteristics vary, dependent on the angle of incidence of the emission to be filtered. The angle of incidence will be unique for each well because each well's specific location is unique relative to the optical filter. Accordingly, all calculations and filter coefficients must be unique per sample well. The multi-component calibration is performed as follows:
- Prior to the real multiplexed (multiple reagent) samples, standards containing only a single reagent in each well are imaged and analyzed. These standards will produce a set of coefficients to be used collectively as multi-component coefficients for each optical filter, for each well. For a given optical filter, the target reagent for that filter should produce the highest output. The other reagents may also have spectra in the filter's bandpass, and will produce smaller outputs, which are a measure of the overlap of those nontarget reagent spectra into the filter signal. For example, the filter's output for the target reagent might be 850, and the filter's output for the other 2 reagents might be 100 and 50, respectively. If the 3 reagents were added together in a single well, the total output would be 1000, and the proportions would be 850:100:50. These coefficients are measured for each well location and filter separately, which gives a complete set of coefficients for simultaneous equations. This will allow a solution for any combination of concentrations of reagent in one sample well. Further in the preferred embodiment, these coefficients will also be normalized by the total intensity read in the “total emission” filter, so that the calculation will result in the same intensity as the instrument would measure if only a single reagent was measured by the “total emission” filter. This calculation may be shown as follows for a simple case of blue and green reagents (abbreviated as R in the calculations), and blue and green and total emission filters (abbreviated as F in the calculations):
-
Let A=(output of the instrument for blue R thru the blue F)/(output of instrument for blue R thru total emission F); -
Let B=(output of the instrument for green R thru the blue F)/(output of instrument for green R thru total emission F); -
Let C=(output of the instrument for blue R thru the green F)/(output of instrument for blue R thru total emission F); -
Let D=(output of the instrument for green R thru the green F)/(output of instrument for green R thru total emission F); - These coefficients are measured for each well prior to running a multi-color run.
Then for a multi-reagent/color run, -
(output of the instrument for the blue F)=A×(true intensity of blue R)+B×(intensity of green R); and -
(output of the instrument for the green F)=C×(true intensity of blue R)+D×(intensity of green R) - These 2 simultaneous equations are then solved for the true intensity of the blue and green reagents by the processing software, under control of
processor 50. - Further in step G, the raw output of the instrument for each filter is normalized for integration time before solving the equations.
- The resulting intensities could then be calibrated as concentration by use of standards as described in the previous section.
- Finally, in step H, the analyzed data is presented in a user-acceptable format, again controlled by
processor 50. - The invention may be further understood by reference to examples of assays practiced in HTS format, demonstrating the dynamic range and flexibility of the NorthStar™ luminometer.
- cAMP standards were serial diluted and added to a 96-well assay plate with alkaline phosphatase conjugated cAMP and anti-cAMP. Plates were processed with the cAMP-Screen™ protocol and imaged for 1 minute on the
NorthStar™ 30 minutes after addition of CSPD®/Sapphire-II™. A sensitivity of 0.06 pM of purified cAMP is achieved with cAMP-Screen™ on the NorthStar™ workstation. The results are depicted inFIG. 8 . - Adrenergic β2 Receptor-expressing C2 cells were plated in a 96-well plate (10,000 cells/well) and stimulated with isoproterenol for 10 minutes. cAMP production was quantitated in cell lysates using the cAMP-Screen™ assay. The assay plate was imaged for 1 minute on the NorthStar™, 30 minutes after addition of CSPD®/Sapphire-II™. Increasing cAMP levels were detected on the NorthStar™ from the stimulated adrenergic receptor. The results are depicted in
FIG. 9 . - pCRE-Luc-Transfected cells were seeded in 96-, 384- and 1,536-well plates, incubated for 20 hours with forskolin, and assayed with the Luc-Screen™ system. PCRE-Luc contains the luciferase reporter gene under the control of a cAMP response element (CRE). Forskolin induces intracellular cAMP production through the irreversible activation of adenylate cyclase. All plate formats demonstrate comparable forskolin-induced cAMP levels. The results are depicted in
FIG. 10 . - pCRE-Luc-Transfected cells were seeded in a 96-well plate. Four random wells were induced for 17 hours with 1 mM forskolin and the entire plate was assayed with the Luc-Screen™ system. The results are shown in
FIG. 11 . - NIH/3T3 cells were co-transfected with pCRE-Luc and pβgal-Control, and seeded into a 96-well microplate (2×104 cells/well). Cells were incubated with forskolin for 17 hours. Modified Dual-Light® Buffer A was added to cells and incubated for 10 minutes. Modified Dual Light® Buffer B was injected and luciferase-catalyzed light emission was measured immediately. Thirty minutes later, Accelerator-II was added, and then β-galactosidase-catalyzed light emission was quantitated on the NorthStar™ HTS workstation. Quantitation is shown graphically in
FIG. 12 . - Fold induction of luciferase activity was calculated following normalization to β-galactosidase activity. The Dual-Light® assay enables the use of a control reporter for normalization, or to monitor non-specific effects on gene expression. This is depicted in
FIG. 13 . - CHO-Aeq-5HT2B cells were loaded with coelenterazine h+/−0.5 μM BAPTA-AM for 4 hours. The antagonist methysergide was added to the charged cells for 30 minutes. 1 μM agonist a-Me-5HT was injected, and the emitted light was integrated for 20 seconds on the NorthStar™ system. The reported IC50 for methysergide (0.6 nM) is unchanged in the presence of BAPTA-AM. The data obtained appears in
FIG. 14 . - CHO-Aeq-OX2-A2 cells (Euroscreen) were loaded with coelenterazine h+/−0.6 μM BAPTA-AM for 4 hours. The peptide agonist Orexin B was injected into the wells, and the emitted light was integrated for 20 seconds on the NorthStar™. Using this assay on the NorthStar™ system, the reported EC50 for Orexin B (0.75 nM) is unchanged in the presence of BAPTA-AM. This is shown in
FIG. 15 . - This invention has been described generically, by reference to specific embodiments and by example. Unless so indicated, no embodiment or example is intended to be limiting. Alternatives will occur to those of ordinary skill in the art without the exercise of inventive skill, and within the scope of the claims set forth below.
Claims (21)
1-13. (canceled)
14. A luminescence detecting apparatus, comprising:
a sample chamber configured to receive a plurality of sampling areas containing respective luminescent samples;
a photosensitive detector;
an optical system configured to produce a sample image of the sampling areas, comprising:
a substrate comprising a two-dimensional array of openings and positioned during use between the plurality of sampling areas and the photosensitive detector, the plurality of sampling areas aligned during use to the array of openings, the array of openings configured to simultaneously pass emissions from at least some of the sample areas to the photosensitive detector and to block some of the emissions from being received by the photosensitive detector; and
a lens disposed along an optical path between the two-dimensional array of openings and the photosensitive detector; and
a processor comprising instructions to a subtract a dark image from the sample image;
wherein the array of openings, the lens, and the processor are together configured to refine values of the sample image.
15. The apparatus of claim 14 , wherein the plurality of sampling areas comprises an array of sample wells.
16. The apparatus of claim 14 , further comprising a defogger configured to prevent condensation.
17. The apparatus of claim 14 , wherein the photosensitive detector is configured to sequentially detect light at different wavelengths emitted from the plurality of sampling areas.
18. The apparatus of claim 14 , further comprising a plurality of luminescent samples disposed within respective ones of the plurality of sample areas during use.
19. The apparatus of claim 18 , wherein each of the plurality of luminescent samples is one of a bioluminescent material or a chemiluminescent material.
20. The apparatus of claim 14 , further comprising a plate, wherein plurality of sampling areas are disposed on the plate.
21. The apparatus of claim 14 , further comprising a filter disposed along the optical path between the two-dimensional array of openings and the detector, the filter configured to permit passage of a specific wavelength of light emitted from the sample areas.
22. The apparatus of claim 14 , wherein the two-dimensional array of openings is in close proximity to the plurality of sampling areas during use, so as to enhance restriction of stray light from the sampling areas.
23. The apparatus of claim 14 , wherein the lens comprises at least one of a Fresnel lens, an aspheric lens, or a molded plastic lens.
24. The apparatus of claim 14 , wherein the photosensitive detector comprises a plurality of photosensitive detector elements.
25. The apparatus of claim 14 , wherein the photosensitive detector comprises a charge coupled device.
26. The apparatus of claim 25 , wherein:
the lens is a primary lens and the apparatus further comprises a camera lens disposed between the primary lens and the charge coupled device; and
the camera lens is configured to simultaneously image the luminescent samples of more than one of sampling areas.
27. The apparatus of claim 14 , wherein the sample chamber comprises a bottom surface and the substrate is positioned during use above the sample chamber.
28. The apparatus of claim 14 , wherein the plurality of sampling areas are in simultaneous optical communication with the photosensitive detector, and wherein the photosensitive detector and the two-dimensional array of openings together are configured to simultaneously detect the plurality of respective luminescent samples contained in the plurality of sampling areas.
29. The apparatus of claim 14 , wherein the sample chamber comprises a bottom surface and the substrate is positioned during use above the sample chamber.
30. A method for analyzing a plurality of luminescent samples, comprising the steps of:
providing a plurality of luminescent samples disposed in a respective plurality of sampling areas;
providing a photosensitive detector;
providing an optical system configured to produce a sample image of the sampling areas, the optical system comprising:
a substrate comprising a two-dimensional array of openings and positioned during use between the plurality of sampling areas and the photosensitive detector, the plurality of sampling areas aligned during use to the array of openings, the array of openings configured to simultaneously pass emissions from at least some of the sample areas to the photosensitive detector and to block some of the emissions from being received by the photosensitive detector; and
a first lens disposed along an optical path between the two-dimensional array of openings and the photosensitive detector; and
placing the plurality of sampling areas in optical communication with the optical system;
passing emissions from at least some of the sampling areas through the two-dimensional array of openings to respective ones of the photosensitive detector;
using the substrate to prevent some of the emissions from the at least some of the sampling areas from being received by the photosensitive detector;
using the photosensitive detector:
producing a dark image; and
producing a sample image
using the array of openings, the lens, and the dark image to produce refined values of the sample image.
31. The apparatus of claim 30 , wherein the photosensitive detector comprises a plurality of photosensitive detector elements.
32. The apparatus of claim 30 , wherein the plurality of sampling areas comprises an array of sample wells.
33. The method of claim 30 , further comprising positioning a second lens disposed along the optical path at a location between the first lens and the photosensitive detector.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190130556A1 (en) * | 2017-10-31 | 2019-05-02 | Kabushiki Kaisha Toshiba | Inspection system and inspection method |
WO2022146981A1 (en) * | 2020-12-31 | 2022-07-07 | Q-State Biosciences, Inc. | Plate imager |
Families Citing this family (73)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU6227900A (en) * | 1999-07-21 | 2001-02-13 | Tropix, Inc. | Luminescence detection workstation |
US6867005B2 (en) * | 2001-10-24 | 2005-03-15 | Beckman Coulter, Inc. | Method and apparatus for increasing the dynamic range and accuracy of binding assays |
JP3875090B2 (en) * | 2001-12-12 | 2007-01-31 | 独立行政法人科学技術振興機構 | Method and apparatus for preventing condensation and freezing of optical glass window |
EP1343001A1 (en) * | 2002-03-04 | 2003-09-10 | Evotec OAI AG | A method for identifying the impacts of interfering effects on secondary light emission data |
US20040174821A1 (en) | 2003-03-04 | 2004-09-09 | Christian Eggeling | Method for detecting the impacts of interfering effects on experimental data |
DE10236029A1 (en) | 2002-08-02 | 2004-02-19 | Cybio Systems Gmbh | Device for dispensing and monitoring the luminescence of individual samples in multi-sample arrangements |
WO2005033712A1 (en) * | 2002-10-29 | 2005-04-14 | E. I. Du Pont De Nemours And Company | Method and apparatus for performing chemical reactions in a plurality of samples |
DE10246481A1 (en) | 2002-09-30 | 2004-04-08 | Cybio Systems Gmbh | Device for calibrating an optical detection channel for the two-dimensional measurement of multi-sample carriers |
CN1695061A (en) | 2002-10-29 | 2005-11-09 | 纳幕尔杜邦公司 | Method and apparatus for performing chemical reactions in a plurality of samples |
US7435602B2 (en) * | 2002-12-20 | 2008-10-14 | Applied Biosystems Inc. | Reducing effects of spectral nonuniformity |
US20040259182A1 (en) * | 2003-06-17 | 2004-12-23 | Brooks Edwards | Arrays for chemiluminescent assays, methods of making the arrays and methods of detecting chemiluminescent emissions on solid supports |
US20050019778A1 (en) * | 2003-07-17 | 2005-01-27 | Voyta John C. | Sequential generation of multiple chemiluminescent signals on solid supports |
US20050026151A1 (en) * | 2003-07-17 | 2005-02-03 | Voyta John C. | Simultaneous generation of multiple chemiluminescent signals on solid supports |
US7233393B2 (en) | 2004-08-05 | 2007-06-19 | Applera Corporation | Signal noise reduction for imaging in biological analysis |
JP3950972B2 (en) * | 2003-11-14 | 2007-08-01 | 国立大学法人名古屋大学 | Bioluminescence measuring device for biological samples |
DE602005007582D1 (en) * | 2004-01-14 | 2008-07-31 | Applera Corp | IN BIOLOGICAL SAMPLES |
US7295316B2 (en) * | 2004-01-14 | 2007-11-13 | Applera Corporation | Fluorescent detector with automatic changing filters |
US8275216B2 (en) * | 2004-06-28 | 2012-09-25 | Inphase Technologies, Inc. | Method and system for equalizing holographic data pages |
US7848595B2 (en) * | 2004-06-28 | 2010-12-07 | Inphase Technologies, Inc. | Processing data pixels in a holographic data storage system |
CN101124725B (en) * | 2004-09-16 | 2012-06-20 | 南方创新国际私人有限公司 | Method and apparatus for resolving individual signals in detector output data |
US8084260B2 (en) * | 2004-11-24 | 2011-12-27 | Applied Biosystems, Llc | Spectral calibration method and system for multiple instruments |
JP4575225B2 (en) * | 2005-04-21 | 2010-11-04 | 株式会社エスジー | Outline inspection device |
JP4789518B2 (en) * | 2005-06-30 | 2011-10-12 | キヤノン株式会社 | Method for detecting target substance using probe-immobilized carrier with manufacturing conditions, and apparatus, kit and system therefor |
KR100817702B1 (en) * | 2005-08-05 | 2008-03-27 | 산요덴키가부시키가이샤 | Reaction Detecting Device |
JP2007046904A (en) * | 2005-08-05 | 2007-02-22 | Sanyo Electric Co Ltd | Reaction detector |
US7630849B2 (en) * | 2005-09-01 | 2009-12-08 | Applied Biosystems, Llc | Method of automated calibration and diagnosis of laboratory instruments |
US20070081920A1 (en) * | 2005-10-12 | 2007-04-12 | Murphy R S | Semi-disposable optoelectronic rapid diagnostic test system |
US20070098596A1 (en) * | 2005-10-14 | 2007-05-03 | University Of South Florida | Handheld microarray reader |
US8968658B2 (en) | 2006-02-08 | 2015-03-03 | Molecular Devices, Llc | Luminescence measurement utilizing cartridge with integrated detector |
US7678330B2 (en) * | 2006-03-01 | 2010-03-16 | Aleksandr Ostrovsky | System, method and apparatus for use in blood testing through luminescence |
US8286578B2 (en) * | 2006-09-18 | 2012-10-16 | Agfa Graphics Nv | Device for coating a peripheral surface of a sleeve body |
JP2008109864A (en) * | 2006-10-30 | 2008-05-15 | Hitachi Ltd | Genetic sequence analysis system |
JP5026851B2 (en) * | 2007-04-23 | 2012-09-19 | 株式会社日立製作所 | Chemiluminescence detector |
EP2160594B1 (en) * | 2007-06-29 | 2011-08-03 | Roche Diagnostics GmbH | Systems and methods for determining cross-talk coefficients in pcr and other data sets |
US8828730B2 (en) * | 2008-08-05 | 2014-09-09 | Synapse B.V. | Method and assembly for measuring thrombin generation in plasma |
US8304251B2 (en) * | 2009-02-18 | 2012-11-06 | Chem Spectra, Inc. | Portable explosive or drug detection system |
US20120107949A1 (en) * | 2009-02-26 | 2012-05-03 | Jeffrey Haas | Test swipe for portable explosive or drug detection system |
US8475717B2 (en) * | 2009-07-07 | 2013-07-02 | Chemspectra, Inc. | Explosive or drug detection reporting system |
WO2011094234A2 (en) * | 2010-01-26 | 2011-08-04 | Georgetown University | Dosimetry system based on optically stimulated luminesence |
US8363887B2 (en) * | 2010-02-03 | 2013-01-29 | Chemspectra, Inc. | Variable fan for portable explosive or drug detection system |
CN202281746U (en) * | 2010-03-06 | 2012-06-20 | 伊鲁米那股份有限公司 | Measuring equipment for detecting optical signal from sample as well as optical module and optical system for measuring equipment |
WO2012073182A1 (en) * | 2010-11-30 | 2012-06-07 | Koninklijke Philips Electronics N.V. | A sensor device for magnetically actuated particles |
JP5591747B2 (en) * | 2011-03-30 | 2014-09-17 | 株式会社日立製作所 | Luminescence measuring device and microorganism counting device |
GB2496315B (en) * | 2011-11-04 | 2014-04-09 | Dynex Technologies Inc | Multiplex optical assembly |
AU2012352965B2 (en) | 2011-12-16 | 2014-08-21 | Li-Cor, Inc. | Luminescence imaging scanner |
KR20130086743A (en) * | 2012-01-26 | 2013-08-05 | 삼성전자주식회사 | Microfluidic device and control method thereof |
CN104303047B (en) * | 2012-03-15 | 2018-01-09 | 生物辐射实验室股份有限公司 | Image for chemiluminescence sample obtains |
US9058648B2 (en) | 2012-03-15 | 2015-06-16 | Bio-Rad Laboratories, Inc. | Image acquisition for chemiluminescent samples |
US9590122B2 (en) * | 2012-05-18 | 2017-03-07 | Siemens Healthcare Diagnostics Inc. | Fish eye lens analyzer |
US9230305B2 (en) * | 2012-12-31 | 2016-01-05 | Nvidia Corporation | Summed area computation using ripmap of partial sums |
DE202013101439U1 (en) | 2013-04-04 | 2013-04-23 | Dynex Technologies Inc. | Optical multiplex arrangement |
JP6449591B2 (en) * | 2013-09-02 | 2019-01-09 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Biological liquid light measuring device |
ITTO20130940A1 (en) | 2013-11-20 | 2015-05-21 | St Microelectronics Srl | KIT FOR BIOCHEMICAL ANALYSIS AND METHOD TO PERFORM A IMPROVED BIOCHEMICAL PROCESS |
CN106661764B (en) | 2014-07-28 | 2020-06-30 | 赛诺菲巴斯德维思设计公司 | Automated imaging and analysis of hemagglutination inhibition assay (HAI) |
JP6496416B2 (en) * | 2015-02-06 | 2019-04-03 | ライフ テクノロジーズ コーポレーション | Method and system for normalizing a pure dye instrument |
BR112017016932B1 (en) * | 2015-02-06 | 2021-04-20 | Life Technologies Corporation | computer-readable non-transient storage method, system and media for identifying a reaction site associated with an amplification curve from a plurality of amplification curves |
CN107889521B (en) * | 2015-02-06 | 2020-03-27 | 生命技术公司 | Method and system for determining an optical region of interest |
US10012590B2 (en) | 2015-02-06 | 2018-07-03 | Life Technolgies Corporation | Methods and systems for biological instrument calibration |
US9867250B1 (en) | 2015-04-20 | 2018-01-09 | The Arizona Board Of Regents On Behalf Of The University Of Arizona | System having a configurable pico-second pulsed LED driver circuit and photomultiplier tube biasing and gating circuits for real-time, time-resolved transient recording of fluorescence |
JP2017067605A (en) * | 2015-09-30 | 2017-04-06 | 高電工業株式会社 | Specimen measurement device and specimen measurement method |
CN109312287A (en) * | 2016-07-29 | 2019-02-05 | 休斯顿大学系统 | The system and method for detecting chemiluminescence reaction |
CN107817227B (en) | 2016-09-12 | 2020-08-28 | 台达电子国际(新加坡)私人有限公司 | Fluorescence detection device |
SG10201609334WA (en) | 2016-11-08 | 2018-06-28 | Delta Electronics Intl Singapore Pte Ltd | Multi-Channel Fluorescence Detection Device |
CN110268108B (en) | 2016-12-12 | 2022-09-06 | 埃克切拉生物科学公司 | Methods and systems for screening using microcapillary arrays |
US10267845B2 (en) * | 2017-05-12 | 2019-04-23 | Delta V Instruments, Inc. | Wafer level burn-in system |
WO2019060373A1 (en) | 2017-09-19 | 2019-03-28 | Beckman Coulter, Inc. | Analog light measuring and photon counting in chemiluminescence measurements |
SG10201801853WA (en) | 2018-03-02 | 2019-10-30 | Delta Electronics Int’L Singapore Pte Ltd | Portable multi-color fluorescence detection device |
WO2021051129A1 (en) * | 2019-09-09 | 2021-03-18 | Cerillo, Llc | A solid-state, multi-well plate reader |
TWI722785B (en) | 2020-01-31 | 2021-03-21 | 台達電子工業股份有限公司 | Optical calibration tool |
US10793772B1 (en) | 2020-03-13 | 2020-10-06 | Accelovant Technologies Corporation | Monolithic phosphor composite for sensing systems |
CN111795808B (en) * | 2020-07-17 | 2022-06-24 | 苏州精濑光电有限公司 | Display screen detection equipment |
US11359976B2 (en) | 2020-10-23 | 2022-06-14 | Accelovant Technologies Corporation | Multipoint surface temperature measurement system and method thereof |
CA3137183C (en) | 2020-11-05 | 2024-02-20 | Accelovant Technologies Corporation | Optoelectronic transducer module for thermographic temperature measurements |
Citations (40)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3895661A (en) * | 1972-08-18 | 1975-07-22 | Pfizer | Cuvette apparatus for testing a number of reactants |
US4003151A (en) * | 1975-10-03 | 1977-01-18 | Dynatech Laboratories, Incorporated | Test plate reader |
US4119850A (en) * | 1977-04-05 | 1978-10-10 | Abbott Laboratories | Multiple sample, radioactive particle counting apparatus |
US4221867A (en) * | 1979-02-02 | 1980-09-09 | Minnesota Mining And Manufacturing Company | Optical microbiological testing apparatus and method |
US4223221A (en) * | 1978-06-19 | 1980-09-16 | Picker Corporation | Scintillation camera uniformity correction |
US4448534A (en) * | 1978-03-30 | 1984-05-15 | American Hospital Corporation | Antibiotic susceptibility testing |
US4498782A (en) * | 1981-05-29 | 1985-02-12 | Science Research Center, Inc. | Assembly for determining light transmissiveness of a fluid |
US4580895A (en) * | 1983-10-28 | 1986-04-08 | Dynatech Laboratories, Incorporated | Sample-scanning photometer |
US4682235A (en) * | 1985-10-18 | 1987-07-21 | Ford Aerospace & Communications Corporation | Ortho-linear imaging device |
US4710031A (en) * | 1985-07-31 | 1987-12-01 | Lancraft, Inc. | Microtiter plate reader |
US4762413A (en) * | 1984-09-07 | 1988-08-09 | Olympus Optical Co., Ltd. | Method and apparatus for measuring immunological reaction with the aid of fluctuation in intensity of scattered light |
US4762420A (en) * | 1986-04-01 | 1988-08-09 | Fisons Plc | Photometric reading device for serological analysis |
US4922092A (en) * | 1986-11-26 | 1990-05-01 | Image Research Limited | High sensitivity optical imaging apparatus |
US5048957A (en) * | 1989-07-11 | 1991-09-17 | Fritz Berthold | Speciman rack with insertable cuvettes |
US5073029A (en) * | 1990-02-16 | 1991-12-17 | Eqm Research, Inc. | Multisource device for photometric analysis and associated chromogens |
US5082628A (en) * | 1989-09-19 | 1992-01-21 | Park Pharmaceuticals, Inc. | Luminometer |
US5112134A (en) * | 1984-03-01 | 1992-05-12 | Molecular Devices Corporation | Single source multi-site photometric measurement system |
US5144136A (en) * | 1989-09-07 | 1992-09-01 | RSM Analytiche Instrumente GmbH | Device for simultaneously measuring particle or quantum beams from many samples at once |
US5151826A (en) * | 1989-10-16 | 1992-09-29 | Combined Optical Industries Limited | Fresnel lens |
US5169601A (en) * | 1990-04-27 | 1992-12-08 | Suzuki Motor Corporation | Immunological agglutination detecting apparatus with separately controlled supplementary light sources |
US5229883A (en) * | 1991-10-28 | 1993-07-20 | Mcdonnell Douglas Corporation | Hybrid binary optics collimation fill optics |
US5290513A (en) * | 1991-07-18 | 1994-03-01 | Laboratorium Prof. Dr. Rudolf Berthold Gmbh & Co. Kg | Radiation measuring device, particularly for luminescence measurements |
US5355215A (en) * | 1992-09-30 | 1994-10-11 | Environmental Research Institute Of Michigan | Method and apparatus for quantitative fluorescence measurements |
US5401465A (en) * | 1992-05-05 | 1995-03-28 | Chiron Corporation | Luminometer with reduced sample crosstalk |
US5426306A (en) * | 1993-10-21 | 1995-06-20 | Associated Universities, Inc. | Fast repetition rate (FRR) fluorometer and method for measuring fluorescence and photosynthetic parameters |
US5578832A (en) * | 1994-09-02 | 1996-11-26 | Affymetrix, Inc. | Method and apparatus for imaging a sample on a device |
US5682232A (en) * | 1995-08-25 | 1997-10-28 | Precision System Science Co., Ltd. | Microplate light-obstruction device and light-emission measuring apparatus |
US5720928A (en) * | 1988-09-15 | 1998-02-24 | New York University | Image processing and analysis of individual nucleic acid molecules |
US5774214A (en) * | 1996-12-12 | 1998-06-30 | Photometrics, Ltd. | Multi-mode imaging apparatus for radiation-emitting or absorbing samples |
US5784152A (en) * | 1995-03-16 | 1998-07-21 | Bio-Rad Laboratories | Tunable excitation and/or tunable detection microplate reader |
US5828067A (en) * | 1993-10-20 | 1998-10-27 | Cambridge Imaging Limited | Imaging method and apparatus |
US5866331A (en) * | 1995-10-20 | 1999-02-02 | University Of Massachusetts | Single molecule detection by in situ hybridization |
US6071748A (en) * | 1997-07-16 | 2000-06-06 | Ljl Biosystems, Inc. | Light detection device |
US6160618A (en) * | 1998-06-19 | 2000-12-12 | Board Of Regents, The University Of Texas System | Hyperspectral slide reader |
US6191852B1 (en) * | 1997-10-14 | 2001-02-20 | Bayer Aktiengesellschaft | Optical measurement system for detecting luminescence or fluorescence signals |
US6246525B1 (en) * | 1998-08-31 | 2001-06-12 | Fuji Photo Film Co., Ltd. | Imaging device |
US6271022B1 (en) * | 1999-03-12 | 2001-08-07 | Biolog, Inc. | Device for incubating and monitoring multiwell assays |
US6473239B2 (en) * | 1998-10-12 | 2002-10-29 | Carl-Zeiss-Stiftung | Imaging system with a cylindrical lens array |
US6608918B1 (en) * | 1996-10-10 | 2003-08-19 | Packard Instrument Company, Inc. | Method and apparatus for assay analysis |
US8278114B2 (en) * | 1999-07-21 | 2012-10-02 | Applied Biosystems, Llc | Method for measuring luminescence at a luminescence detection workstation |
Family Cites Families (80)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3264474A (en) * | 1963-09-09 | 1966-08-02 | American Instr Co Inc | Phosphorimeter attachment for fluorometer |
US3678564A (en) * | 1968-01-30 | 1972-07-25 | Brunswick Corp | Method of producing high resolution images and structure for use therein |
US3628028A (en) * | 1968-03-01 | 1971-12-14 | Honeywell Inc | Window cleaning apparatus for photometric instruments |
US3581308A (en) * | 1969-04-11 | 1971-05-25 | Joseph T Mcnaney | Light guide character forming mask and display device control element |
US3668395A (en) * | 1969-12-17 | 1972-06-06 | Nuclear Chicago Corp | Scintillation camera having improved peripheral response |
US3723746A (en) * | 1970-01-07 | 1973-03-27 | Nat Res Dev | Fire detecting apparatus sensitive to refraction |
US3745359A (en) * | 1971-03-22 | 1973-07-10 | Picker Corp | Scintillation crystal with reflection inhibiting material and scintillation device embodying the crystal |
US3754866A (en) * | 1971-07-30 | 1973-08-28 | Sherwood Medical Ind Inc | Optical detecting system |
US4056724A (en) * | 1975-02-27 | 1977-11-01 | International Diagnostic Technology | Fluorometric system, method and test article |
US4004150A (en) * | 1975-05-01 | 1977-01-18 | Samuel Natelson | Analytical multiple component readout system |
US4099920A (en) * | 1977-03-17 | 1978-07-11 | Baxter Travenol Laboratories, Inc. | Temperature control and stirring apparatus for luminescence measuring photometer |
US4126783A (en) * | 1977-05-25 | 1978-11-21 | Butler-Newton, Inc. | Radiation imaging system |
ZA783198B (en) * | 1978-06-05 | 1979-09-26 | Sphere Invest | Improvements relating to sorting systems |
JPS55500714A (en) | 1978-06-14 | 1980-10-02 | ||
US4190368A (en) * | 1978-06-19 | 1980-02-26 | Monitor Labs, Inc. | Sulfur monitor analyzer |
EP0025350A3 (en) | 1979-09-05 | 1981-06-10 | Dynatech Ag | Apparatus for detecting luminescent reactions |
US4307294A (en) * | 1980-03-04 | 1981-12-22 | Campbell Duncan B | Electro-mechanical control means for space communication receiver |
US4437160A (en) * | 1980-07-31 | 1984-03-13 | Blum Alvin S | Photon emission imaging apparatus and method |
SE423458B (en) * | 1980-09-10 | 1982-05-03 | Agne Larsson | DEVICE OF A CAMERA INCLUDING A DIFFERENT COLLIMATOR |
US4392746A (en) * | 1980-12-08 | 1983-07-12 | Portalab Instruments Limited | Portable photometer |
US4521689A (en) * | 1983-02-24 | 1985-06-04 | General Electric Company | Modular radiation-detecting array |
US4695729A (en) * | 1983-07-19 | 1987-09-22 | Fuji Electric Co., Ltd. | Tubular part wall thickness measuring device |
US4968148A (en) * | 1984-03-01 | 1990-11-06 | Molecular Devices Corporation | Single source multi-site photometric measurement system |
JPS61122601A (en) * | 1984-11-19 | 1986-06-10 | Canon Inc | Fresnel lens |
US4772453A (en) * | 1985-03-01 | 1988-09-20 | Lisenbee Wayne F | Luminiscence measurement arrangement |
US5656493A (en) * | 1985-03-28 | 1997-08-12 | The Perkin-Elmer Corporation | System for automated performance of the polymerase chain reaction |
US4949079A (en) * | 1985-04-19 | 1990-08-14 | Hugh Loebner | Brightpen/pad graphic device for computer inputs and the like |
US5104621A (en) * | 1986-03-26 | 1992-04-14 | Beckman Instruments, Inc. | Automated multi-purpose analytical chemistry processing center and laboratory work station |
JPH07117502B2 (en) * | 1986-11-25 | 1995-12-18 | ペトロ−カナダ・インコ−ポレ−テツド | Measuring device |
US4810348A (en) * | 1987-03-16 | 1989-03-07 | Helena Laboratories Corporation | Automatic electrophoresis apparatus and method |
US4986891A (en) * | 1987-03-16 | 1991-01-22 | Helena Laboratories Corporation | Automatic electrophoresis apparatus and method |
US5147522A (en) * | 1987-03-16 | 1992-09-15 | Helena Laboratories Corporation | Automatic electrophoresis apparatus and method |
WO1990002326A1 (en) * | 1988-08-23 | 1990-03-08 | Bio-Mediq (Australia) Pty. Ltd. | Optical fluid analysis imaging and positioning |
US4967084A (en) * | 1989-02-02 | 1990-10-30 | The University Of Michigan | Multi-sample scintillation counter using position-sensitive detector |
JPH0620458B2 (en) * | 1989-03-14 | 1994-03-23 | 新技術事業団 | High directivity imaging element and high directivity imaging device |
JP2750605B2 (en) * | 1989-05-17 | 1998-05-13 | スズキ株式会社 | Particle aggregation pattern determination method |
FI895615A (en) * | 1989-11-23 | 1991-05-24 | Valtion Teknillinen | MAETNINGSANORDNING FOER MAETNING AV FEL I ROER. |
JPH0678978B2 (en) * | 1990-05-25 | 1994-10-05 | スズキ株式会社 | Aggregation pattern detector |
JPH05231938A (en) * | 1991-02-07 | 1993-09-07 | Res Dev Corp Of Japan | Highly sensitive multiwavelength spectral apparatus |
DE4139368C2 (en) * | 1991-11-29 | 1996-07-11 | Berthold Lab Prof Dr | Device for measuring the radioactivity distribution on a flat sample |
JPH05157684A (en) * | 1991-12-02 | 1993-06-25 | Seikagaku Kogyo Co Ltd | Absorptionmeter |
US5315375A (en) * | 1992-02-11 | 1994-05-24 | Acrogen, Inc. | Sensitive light detection system |
US5329461A (en) * | 1992-07-23 | 1994-07-12 | Acrogen, Inc. | Digital analyte detection system |
US5674698A (en) * | 1992-09-14 | 1997-10-07 | Sri International | Up-converting reporters for biological and other assays using laser excitation techniques |
GB9223259D0 (en) * | 1992-11-06 | 1992-12-23 | Quatro Biosystems Ltd | Method and apparatus for image analysis |
WO1995004264A1 (en) * | 1993-07-29 | 1995-02-09 | Wesley-Jessen Corporation | Inspection system for optical components |
US5766875A (en) * | 1993-07-30 | 1998-06-16 | Molecular Devices Corporation | Metabolic monitoring of cells in a microplate reader |
US5436718A (en) * | 1993-07-30 | 1995-07-25 | Biolumin Corporation | Mutli-functional photometer with movable linkage for routing optical fibers |
US5946431A (en) * | 1993-07-30 | 1999-08-31 | Molecular Dynamics | Multi-functional photometer with movable linkage for routing light-transmitting paths using reflective surfaces |
CA2129787A1 (en) * | 1993-08-27 | 1995-02-28 | Russell G. Higuchi | Monitoring multiple amplification reactions simultaneously and analyzing same |
US5961926A (en) * | 1993-09-27 | 1999-10-05 | Packard Instrument Co., Inc. | Microplate assembly and method of preparing samples for analysis in a microplate assembly |
JPH0926426A (en) * | 1995-07-12 | 1997-01-28 | Hamamatsu Photonics Kk | Photometer |
CA2157755A1 (en) * | 1995-09-07 | 1997-03-08 | Peter Ramm | Camera system for imaging at low light levels |
US5611994A (en) * | 1995-11-22 | 1997-03-18 | Dynatech Laboratories, Inc. | Luminometer |
US5751444A (en) * | 1995-12-18 | 1998-05-12 | Adobe Systems Incorporated | Imaging apparatus for copying bound documents |
US6602657B1 (en) | 1995-12-28 | 2003-08-05 | Tropix, Inc. | Multiple reporter gene assay |
US5657118A (en) * | 1996-01-23 | 1997-08-12 | Lee; John T. S. | Device and method for detection/measurement of light |
US5567294A (en) * | 1996-01-30 | 1996-10-22 | Board Of Governors, University Of Alberta | Multiple capillary biochemical analyzer with barrier member |
CA2263226C (en) * | 1996-08-16 | 2006-10-10 | Imaging Research, Inc. | A digital imaging system for assays in well plates, gels and blots |
JP3182527B2 (en) * | 1996-09-03 | 2001-07-03 | 株式会社ヤトロン | Chemiluminescence measurement method and kit |
US5798263A (en) * | 1996-09-05 | 1998-08-25 | Promega Corporation | Apparatus for quantifying dual-luminescent reporter assays |
US5854684A (en) * | 1996-09-26 | 1998-12-29 | Sarnoff Corporation | Massively parallel detection |
JPH10170444A (en) * | 1996-12-06 | 1998-06-26 | Hamamatsu Photonics Kk | Light-measuring apparatus |
IL129767A0 (en) * | 1996-12-12 | 2000-02-29 | Prolume Ltd | Apparatus and method for detecting and identifying infectious agents |
JPH10197449A (en) * | 1997-01-07 | 1998-07-31 | Hamamatsu Photonics Kk | Light measuring apparatus |
US5985214A (en) * | 1997-05-16 | 1999-11-16 | Aurora Biosciences Corporation | Systems and methods for rapidly identifying useful chemicals in liquid samples |
FR2766410A1 (en) | 1997-07-11 | 1999-01-29 | Investix Sa | THERMAL PRINTING MECHANISM |
WO1999008233A1 (en) * | 1997-08-07 | 1999-02-18 | Imaging Research Inc. | A digital imaging system for assays in well plates, gels and blots |
US6043506A (en) * | 1997-08-13 | 2000-03-28 | Bio-Rad Laboratories, Inc. | Multi parameter scanner |
DE19748211A1 (en) * | 1997-10-31 | 1999-05-06 | Zeiss Carl Fa | Optical array system and reader for microtiter plates |
US5939024A (en) * | 1997-12-23 | 1999-08-17 | Packard Instrument Co. | Microplate assembly |
US6198577B1 (en) * | 1998-03-10 | 2001-03-06 | Glaxo Wellcome, Inc. | Doubly telecentric lens and imaging system for multiwell plates |
US6057163A (en) * | 1998-04-28 | 2000-05-02 | Turner Designs | Luminescence and fluorescence quantitation system |
EP3093649B1 (en) * | 1998-05-16 | 2019-05-08 | Life Technologies Corporation | A combination of a reaction apparatus and an optical instrument monitoring dna polymerase chain reactions |
US6022812A (en) | 1998-07-07 | 2000-02-08 | Alliedsignal Inc. | Vapor deposition routes to nanoporous silica |
US7387891B2 (en) | 1999-05-17 | 2008-06-17 | Applera Corporation | Optical instrument including excitation source |
US6238944B1 (en) * | 1999-12-21 | 2001-05-29 | Xerox Corporation | Buried heterostructure vertical-cavity surface-emitting laser diodes using impurity induced layer disordering (IILD) via a buried impurity source |
US7435602B2 (en) * | 2002-12-20 | 2008-10-14 | Applied Biosystems Inc. | Reducing effects of spectral nonuniformity |
US7295316B2 (en) * | 2004-01-14 | 2007-11-13 | Applera Corporation | Fluorescent detector with automatic changing filters |
JP5157684B2 (en) | 2008-06-30 | 2013-03-06 | 日本軽金属株式会社 | Hypereutectic Al-Si alloy casting method and ingot |
-
2000
- 2000-07-21 AU AU62279/00A patent/AU6227900A/en not_active Abandoned
- 2000-07-21 EP EP00948839A patent/EP1221038A4/en not_active Ceased
- 2000-07-21 JP JP2001512276A patent/JP5079958B2/en not_active Expired - Lifetime
- 2000-07-21 CA CA002380307A patent/CA2380307A1/en not_active Abandoned
- 2000-07-21 US US09/621,961 patent/US6518068B1/en not_active Expired - Lifetime
- 2000-07-21 WO PCT/US2000/019845 patent/WO2001007896A1/en active Application Filing
-
2002
- 2002-12-20 US US10/323,669 patent/US20030092194A1/en not_active Abandoned
-
2005
- 2005-10-18 US US11/251,873 patent/US7670848B2/en not_active Expired - Lifetime
-
2010
- 2010-03-02 US US12/716,219 patent/US8278114B2/en not_active Expired - Lifetime
-
2012
- 2012-06-19 JP JP2012137473A patent/JP5551211B2/en not_active Expired - Lifetime
- 2012-08-13 US US13/584,628 patent/US8865473B2/en not_active Expired - Fee Related
-
2013
- 2013-12-27 JP JP2013272701A patent/JP6059132B2/en not_active Expired - Lifetime
-
2014
- 2014-10-15 US US14/515,433 patent/US20150080256A1/en not_active Abandoned
Patent Citations (41)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3895661A (en) * | 1972-08-18 | 1975-07-22 | Pfizer | Cuvette apparatus for testing a number of reactants |
US4003151A (en) * | 1975-10-03 | 1977-01-18 | Dynatech Laboratories, Incorporated | Test plate reader |
US4119850A (en) * | 1977-04-05 | 1978-10-10 | Abbott Laboratories | Multiple sample, radioactive particle counting apparatus |
US4448534A (en) * | 1978-03-30 | 1984-05-15 | American Hospital Corporation | Antibiotic susceptibility testing |
US4223221A (en) * | 1978-06-19 | 1980-09-16 | Picker Corporation | Scintillation camera uniformity correction |
US4221867A (en) * | 1979-02-02 | 1980-09-09 | Minnesota Mining And Manufacturing Company | Optical microbiological testing apparatus and method |
US4498782A (en) * | 1981-05-29 | 1985-02-12 | Science Research Center, Inc. | Assembly for determining light transmissiveness of a fluid |
US4580895A (en) * | 1983-10-28 | 1986-04-08 | Dynatech Laboratories, Incorporated | Sample-scanning photometer |
US5112134A (en) * | 1984-03-01 | 1992-05-12 | Molecular Devices Corporation | Single source multi-site photometric measurement system |
US4762413A (en) * | 1984-09-07 | 1988-08-09 | Olympus Optical Co., Ltd. | Method and apparatus for measuring immunological reaction with the aid of fluctuation in intensity of scattered light |
US4710031A (en) * | 1985-07-31 | 1987-12-01 | Lancraft, Inc. | Microtiter plate reader |
US4682235A (en) * | 1985-10-18 | 1987-07-21 | Ford Aerospace & Communications Corporation | Ortho-linear imaging device |
US4762420A (en) * | 1986-04-01 | 1988-08-09 | Fisons Plc | Photometric reading device for serological analysis |
US4922092A (en) * | 1986-11-26 | 1990-05-01 | Image Research Limited | High sensitivity optical imaging apparatus |
US5720928A (en) * | 1988-09-15 | 1998-02-24 | New York University | Image processing and analysis of individual nucleic acid molecules |
US5048957A (en) * | 1989-07-11 | 1991-09-17 | Fritz Berthold | Speciman rack with insertable cuvettes |
US5144136A (en) * | 1989-09-07 | 1992-09-01 | RSM Analytiche Instrumente GmbH | Device for simultaneously measuring particle or quantum beams from many samples at once |
US5082628A (en) * | 1989-09-19 | 1992-01-21 | Park Pharmaceuticals, Inc. | Luminometer |
US5151826A (en) * | 1989-10-16 | 1992-09-29 | Combined Optical Industries Limited | Fresnel lens |
US5073029A (en) * | 1990-02-16 | 1991-12-17 | Eqm Research, Inc. | Multisource device for photometric analysis and associated chromogens |
US5169601A (en) * | 1990-04-27 | 1992-12-08 | Suzuki Motor Corporation | Immunological agglutination detecting apparatus with separately controlled supplementary light sources |
US5290513A (en) * | 1991-07-18 | 1994-03-01 | Laboratorium Prof. Dr. Rudolf Berthold Gmbh & Co. Kg | Radiation measuring device, particularly for luminescence measurements |
US5229883A (en) * | 1991-10-28 | 1993-07-20 | Mcdonnell Douglas Corporation | Hybrid binary optics collimation fill optics |
US5401465A (en) * | 1992-05-05 | 1995-03-28 | Chiron Corporation | Luminometer with reduced sample crosstalk |
US5355215A (en) * | 1992-09-30 | 1994-10-11 | Environmental Research Institute Of Michigan | Method and apparatus for quantitative fluorescence measurements |
US5828067A (en) * | 1993-10-20 | 1998-10-27 | Cambridge Imaging Limited | Imaging method and apparatus |
US5426306A (en) * | 1993-10-21 | 1995-06-20 | Associated Universities, Inc. | Fast repetition rate (FRR) fluorometer and method for measuring fluorescence and photosynthetic parameters |
US5578832A (en) * | 1994-09-02 | 1996-11-26 | Affymetrix, Inc. | Method and apparatus for imaging a sample on a device |
US5784152A (en) * | 1995-03-16 | 1998-07-21 | Bio-Rad Laboratories | Tunable excitation and/or tunable detection microplate reader |
US5682232A (en) * | 1995-08-25 | 1997-10-28 | Precision System Science Co., Ltd. | Microplate light-obstruction device and light-emission measuring apparatus |
US5866331A (en) * | 1995-10-20 | 1999-02-02 | University Of Massachusetts | Single molecule detection by in situ hybridization |
US6608918B1 (en) * | 1996-10-10 | 2003-08-19 | Packard Instrument Company, Inc. | Method and apparatus for assay analysis |
US5774214A (en) * | 1996-12-12 | 1998-06-30 | Photometrics, Ltd. | Multi-mode imaging apparatus for radiation-emitting or absorbing samples |
US6071748A (en) * | 1997-07-16 | 2000-06-06 | Ljl Biosystems, Inc. | Light detection device |
US6191852B1 (en) * | 1997-10-14 | 2001-02-20 | Bayer Aktiengesellschaft | Optical measurement system for detecting luminescence or fluorescence signals |
US6160618A (en) * | 1998-06-19 | 2000-12-12 | Board Of Regents, The University Of Texas System | Hyperspectral slide reader |
US6246525B1 (en) * | 1998-08-31 | 2001-06-12 | Fuji Photo Film Co., Ltd. | Imaging device |
US6473239B2 (en) * | 1998-10-12 | 2002-10-29 | Carl-Zeiss-Stiftung | Imaging system with a cylindrical lens array |
US6271022B1 (en) * | 1999-03-12 | 2001-08-07 | Biolog, Inc. | Device for incubating and monitoring multiwell assays |
US8278114B2 (en) * | 1999-07-21 | 2012-10-02 | Applied Biosystems, Llc | Method for measuring luminescence at a luminescence detection workstation |
US8865473B2 (en) * | 1999-07-21 | 2014-10-21 | Applied Biosystems, Llc | Luminescence detecting apparatuses and methods |
Non-Patent Citations (3)
Title |
---|
Akhavan-Tafti, H. et al, Journal or Bioluminescence and Chemiluminescence 1994, 9, 155-164. * |
Fowler, A. et al, Genetic Engineering News 1998, 18, 34. * |
Neri, D. et al, BioTechniques 1996, 20, 708-713. * |
Cited By (5)
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US20190130556A1 (en) * | 2017-10-31 | 2019-05-02 | Kabushiki Kaisha Toshiba | Inspection system and inspection method |
US10902578B2 (en) * | 2017-10-31 | 2021-01-26 | Kabushiki Kaisha Toshiba | Inspection system and inspection method |
US20210110530A1 (en) * | 2017-10-31 | 2021-04-15 | Kabushiki Kaisha Toshiba | Inspection system and inspection method |
US11734811B2 (en) * | 2017-10-31 | 2023-08-22 | Kabushiki Kaisha Toshiba | Inspection system and inspection method |
WO2022146981A1 (en) * | 2020-12-31 | 2022-07-07 | Q-State Biosciences, Inc. | Plate imager |
Also Published As
Publication number | Publication date |
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US8865473B2 (en) | 2014-10-21 |
US20060088444A1 (en) | 2006-04-27 |
JP6059132B2 (en) | 2017-01-11 |
US20030092194A1 (en) | 2003-05-15 |
JP5079958B2 (en) | 2012-11-21 |
EP1221038A1 (en) | 2002-07-10 |
US7670848B2 (en) | 2010-03-02 |
AU6227900A (en) | 2001-02-13 |
US20120309103A1 (en) | 2012-12-06 |
CA2380307A1 (en) | 2001-02-01 |
JP2014098709A (en) | 2014-05-29 |
EP1221038A4 (en) | 2004-09-08 |
JP5551211B2 (en) | 2014-07-16 |
US6518068B1 (en) | 2003-02-11 |
US8278114B2 (en) | 2012-10-02 |
JP2003505691A (en) | 2003-02-12 |
WO2001007896A1 (en) | 2001-02-01 |
US20100248387A1 (en) | 2010-09-30 |
JP2012230110A (en) | 2012-11-22 |
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