CA2050762A1 - Flow imaging cytometer - Google Patents
Flow imaging cytometerInfo
- Publication number
- CA2050762A1 CA2050762A1 CA002050762A CA2050762A CA2050762A1 CA 2050762 A1 CA2050762 A1 CA 2050762A1 CA 002050762 A CA002050762 A CA 002050762A CA 2050762 A CA2050762 A CA 2050762A CA 2050762 A1 CA2050762 A1 CA 2050762A1
- Authority
- CA
- Canada
- Prior art keywords
- image capturing
- image
- light
- light source
- flow
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000003384 imaging method Methods 0.000 title claims abstract description 23
- 239000002245 particle Substances 0.000 claims abstract description 8
- 230000001678 irradiating effect Effects 0.000 claims description 15
- 238000001514 detection method Methods 0.000 claims description 5
- 230000000873 masking effect Effects 0.000 claims description 4
- 230000005284 excitation Effects 0.000 abstract description 6
- 238000012544 monitoring process Methods 0.000 abstract description 4
- 238000000034 method Methods 0.000 description 5
- 239000004065 semiconductor Substances 0.000 description 5
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 235000018734 Sambucus australis Nutrition 0.000 description 1
- 244000180577 Sambucus australis Species 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G01N15/1433—
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1425—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its control arrangement
- G01N15/1427—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its control arrangement with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
- G01N15/1459—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1404—Fluid conditioning in flow cytometers, e.g. flow cells; Supply; Control of flow
- G01N2015/1413—Hydrodynamic focussing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
- G01N2015/144—Imaging characterised by its optical setup
Abstract
FLOW IMAGING CYTOMETER
ABSTRACT OF THE DISCLOSURE:
An imaging flow cytometer is provided with a continuous-emission light source for continuously monitoring cells passing through the image capturing area of a video camera for cell-image capturing, and with an excitation light source for picking up a fluorescent image of a cell.
When a line sensor monitoring cell passage through the cytometer senses such cell passage, the cell is irradiated with strobe light and then, after waiting for the cell to move a fixed distance, with the excitation light. Thus, a particle analyzer is provided in which an image by white light resulting from the strobe light and a fluorescent image resulting from the excitation light can be captured simultaneously by a single video camera in either upper and lower halves or right and left halves of one imaged frame.
ABSTRACT OF THE DISCLOSURE:
An imaging flow cytometer is provided with a continuous-emission light source for continuously monitoring cells passing through the image capturing area of a video camera for cell-image capturing, and with an excitation light source for picking up a fluorescent image of a cell.
When a line sensor monitoring cell passage through the cytometer senses such cell passage, the cell is irradiated with strobe light and then, after waiting for the cell to move a fixed distance, with the excitation light. Thus, a particle analyzer is provided in which an image by white light resulting from the strobe light and a fluorescent image resulting from the excitation light can be captured simultaneously by a single video camera in either upper and lower halves or right and left halves of one imaged frame.
Description
2~7~
--I
FLOW IMAGING CYTOMETER
BACKGROUND OF THE INVENTION:
1. Field of the Invention _ This invention relates to a ~low imaging cytometer.
More particularly, the invention relates to a flow-ima~in~
particle analyzing system in which cells fluorescently stained in a manner suitable for the particular cells o~
interest are introduced to a ~low cell to be ~ormed into a planar sheathed flow and irradiated with white light ~strobe light) to obtain an image by white light and e~cited with l~ser light to obtain a ~luorescent image in a highly efficient manner, and in which the two types of images can be captured simultaneously by a sin~le video camera and subject to analysis.
2. Description of the Prior Art -Attempts have been made to irradiate cells, wh:ich have been stained and smeared on a glass slide, with :Light such as risible light or ultraviolet light under a micro-scope, capture a ~luorescent image o~ cells o~ interest, analyze the resulting image and obtain physiolo~ical in~ormation relating -to the cells. However, a method o~
this ~ind is not suited to the analytical processing of a la~ge number of ceIls in a short time, and analysis using fluorescen~ lmages has only limited application.
In another example of the conventionaI flow cytometer, the cell information is obtained using a gross value o~ the fluorescence emitted from the fluorescently stained cell. In other words, the fluorescence emitted ~rom each portion o~ the cell is integrated over the entirety of the cell, and the cell information is obtained in the form of such an integrated value. Though such a method Iends i~sel~ ~o analysis of a large number of cells in a short period of time, it is not possi~le to acquire detailed information as to which portions o~ individual cells have been stained and caused to emit ~luorescence.
Consequently, this method is limited in terms of analytical performance.
2~7~
_ 7 _ On the other hand, a cell classi~ying apparatus ~hat has been put into practical use employs a technique in which cells stained in a manner suitable for a particular cell of interest are i.ntroduced to a ~low cell to be formed into a planar sheathed ~low and irradia-ted with strobe light, a still picture is ob-tained b~ a ~ideo camera and image processing is applied. However, the state of the art is such that the capturing and analysis of fluorescent images o~ individual cells using this method have still not reached a practical sta~e because o~ problems related to fluorescent i.maging sensitivity. The present invention makes use of the technology employed in a flow imaging cytometer of the type having a high image capturin~
efficiency, as previously proposed in the specification of Japanese Patent Application ~o. 185794/199O.
SUMMARY OF THE INVENTION:
Thus, the art still lacks a definitive ~low-imaging particle analyzing system Por sensing cells that pass through an image capturing area.and irradiating the cells with concentrated exciting light,. thereby to assure the required fluorescent intensity and obtain a fluorescent image, a~d for subjecting the fluorescent image, as well as a~ image by white light of the cells derived from the conventional white-light source, to hi~hly ef~icient image capturing and analysis using a single video camera.
Accordingly, an object of the present invention is to pro~ide a ~low imaging cytometer which e~pands upon the idea of the previously proposed (the aforementioned Japanese Patent Application No. 185794/1990. hereinafter referred to as "the earlier application'i) flow imaging cytometer of the type having a high image capturing efficiency, wherein fluorescence emitted by a fluorescently stained cell is obtained as a two-dimensional image at the same time as an image.by white light acquired by conventional strobe-light (white-light) irradiation.
According to the present invention, the foregoing object is attained by providing a flow imaging cytometer comprising a flow cell formed to include a flat flow -3- ~ 7~2 path f'or causing a specimen solution containing p~rticle components to be sensed ~o flow as a ~lat stream, ~irs~
and third light sources arranged on a ~irst side of the ~`low cell for irradiating the specimen solution in the flow cell with pulsed light, ~irst image capturing means arranged on an opposite side of the flow cell for picking up still pictures of the particle components in the specimen solution irradiated by the ~irst and third light s;ources, a second light source arranged on the eirst side o~ the ~low cell for irradiating the specimen solution in the flow cell with light continuously, second image capturing means arranged on the opposite side of the flow cell for picking up an image of the specimen solution irradiated by the second light source, processing means for executing prescribed analysis based upon image data ~rom the ~i.rst and seco:nd image capturing means, and control means ~or detecting the particle components based upon thè image data ~rom the second image capturlng means, and on the basis o~ such detection, ~or causing the third light source to emit light ~irst, ~ollowed by the first li~ht source upon passage of a prescribed time, within an image capturing period o~ the first image capturing means, wherein the first light source is a light source ~or e~citing fluorescence, the third light source ls a light source ~or emitting white light, and the image resultlng from the ~irst light source and the image resulting ~rom the third light source are each captured in a di~erent area on a light-detecting surface o~ the ~irst image capturing means.
The flow imaging cytometer o~ the present invention is further characterized in that the first image capturing means has a two-dimensional image capturing area on the ~low o~ the specimen solu~ion, the second image capturing means has a linear ima~e capturing area on ~he ~low o-~ the specimen solution, the image capturing area o~ the second image capturing means is ~ormed so as to cross the flow o~
the specimen solution within the image capturing area o~
the first image capturing means, the image capturing area o~ the ~irst image capturing means is divided into a zone 2~762 which includes, and a zone which does not include, the image capturing area of the second image capturing ~eans, and an image in one of these zones resulting from irradiation by the third light source and an image in the o~her o~ these zones resulting ~rom irradiation by the ~irst light source are captured by the first image capturing means.
The flow imaging cytometer of the present invention is further characterized by having masking means for masking light irradiating the ~irst image capturlng means in such a manner that the two ima~es do not overlap each other on the light-detecting surface of the first image capturing means.
The flow imaging cytometer of the present invention is further characterized by having means for forming the irradiating light ~rom the -first llght source into an elliptical shape, and in that the light-detectin~ system o~ a fluorescent image is provided with an image intensifier operated only when the ~luorescent image is captured.
Other features and advantages of the present invention will be apparent from the ~ollowing description taken in conjunction with the accompanying drawi~gs.
BRIEF DESCRIPTION OF THE DRAWINGS:
Fig. 1 is a block diagram illustrating the construction of a ~low imaging cytometer according to the present invention;
Fig. 2 is an e~planatory view illustrating an example o~ a light-irradiating area and an image capturing area of a flow~cell;
Fig. 3 is a timing chart illustrating irradiation timing and the timing of a gating signal for an image intensifier;
Fig. 4 is a diagram showing an example of an imaged frame of a cell captured by a video camera; and Fig. 5 is a diagram showing e.~amples of semicircular and rectangular masks.
35 DETAILED DESCRIPTION OF THE PREFERR_ EMBODI~lENTS:
A preferred embodiment of a flow imaging cytometer according to the present invention will now be described with reference to the drawings. The flow imaging cytometer ' ' ' . ' :' ' .
. . ' ' :' . ~; - :
. .
2~7~2 ;, includes, in addition to the light source (a near in-~rared semiconductor laser) and line sensor for monitoring passage o~ cells in the earlier application, an excitation light source for obtainin~ a fluorescent image~ an image in~en-sifier for intensifying the fluorescence, and variousmirrors, filters and masks e~ployed so that the fluorescent image as well as a conventional image by white light can be acquired by a single video camera.
As shown in Fig. 1, the flow imaging cytometer o~
the invention includes a planar-sheath flow cell 6 to which a specimen solution containing stained cells is introduced.
In order that passage of these cells through an image cap-turing area of a video camera 24 may be monitored at all times, the image capturing area is irradiated continuously with laser light -~rom a near ln~rared semiconductor laser 45. The light ~rom the semiconductor laser 45 is colli-mated by a collimator lens 46, and the collimated light is reflected by a dichroic mirror 4 upon passing through a cylindrical lens 47. The reflected light is stopped down to a finely elongated beam spot perpendicular to the direction of cell move by a condenser lens 5 and irradiates an image capturing area o~ a line sensor 14, as illustrated in Fig. 2. In this embodiment, the image capturing area of the line sensor 14 is provided slightly above mid-center of the upper half of the image capturing area of video camera 24.
The light from the semiconductor laser 45 leaving the image capturing area passes through an objective lens 8 a~d is then split by a beam-splitter il. Part of the light ~rom the beam-splitter 11 passes through a dichroic mirro~
;12 and enters to a projecting lens 13, which proceeds to Yorm an imaFe on the line sensor 14. The line sensor 14 successively produces voltage outputs conforming to the accumulated amount of photoelectrical conversion of each pixel s~posed for a scannin~ cycle (several tens of microseconds) o~ one line. By means of signal processing similar to that set forth in -the earlier application, a trigger for strobe-light irradiat~on is applied when :
2 ~ 2 C~
a cell crosses the image cap~uring area of the line sensor 1~ during e~en-numbered field interYals of the ~ideo camera 24.
The processing time from the instant a cell crosses the image capturing area of the line sensor 14 until a strobe 1 is triggered is lOo - 200 ~sec î~ the scanning cycle of the line sensor 14 is 5~ ~sec. On the assumption that the ~low velocity of cells in flow cell 6 is 30 ~m/sec, a cell will move 3 - 6 ~m in this period o~ time. Accord-ingly, the image of a cell obtained by being irradiated withthe strobe 1 will always fall in the area located in the upper half o~ one imaged ~rame, as illustrated in Fig. 4.
The strobe light from strobe 1 is collimated by a collimator lens 9, the collimated light passes through a condenser lens 10 and dichroic mirrors 4, 4A and enters to the condenser lens 5, by virtue o~ which the entirety o~
the image capturing area of video camera 24 is irradlated with the strobe light substantially uniPormly. This strobe light which has passed through the image capturing area is reflected by the dichroic mirror 12 upon being acted upon by the objective lens 8 and beam-splitter 11. The reflected light has its near infrared component cut by a ~ilter 20A, with the resulting light entering to a projecting lens 15.
The latter forms an image upon a semicircular mask 16. shown in Fi~. 5. The lower hal~ of the image is blocked by the . mask 16, as a result of which an image is ~ormed on only hal~ o~ a CCD area sensor of the video camera 24 via a rela~
lens 27 and half-mirror 19.
The part of the light reflected by the beam-splitter 11 passes through a filter 20 and a projecting lens 21, whereby an image is formed on the photoelectric surface of image~intensifier 22. Since gating is applied in such a manner that a voltage is not impressed upon the image intensifier 22 at this poin~ in time, an image does not appear on its output sur~ace. A gating signal ~or this purpose is produced by a discriminator/controller 28, which i5 ~or judging when a cell has passed through the ima~e capturing area, and for controlling the light sources.
.
.
7 ~ 2 ~ ter a cell has been irradiated with light from the strobe 1, the system wai.ts for the cell to travel to a position in the lower half of the image ca~turing area before irradiating the cell with light from an e~citation light source 35 (for example, an He-Cd laser or xenon lamp).
A trigger signal for this purpose is produced by the light-source controller 28. The light from the light source 35 is rendered into an oblong form by a cylindrical lens 36, and the light ~rom lens 36 is re~lected by the dichroic mirror 4A. The reflected light is stopped down to a ~inely elongated beam spot perpendicular to the direction of cell move by the condenser lens 5 and irradiates the mid-center region o~ the lower half of the image capturing area o~
video camera 24t as depicted in Fig. 2. The cell will be moving through this irradiated area at this time. If the moving speed of the cell through the flow cell 6 is 30 mm~sec and the image capturing area o~ the video camera 24 has a size of 150 ~ 150 ~m, then control should be exer-cised in such a manner that the image capturing area is irradlated with the exciting light approximately 2.5 msec a~ter this area has been irradiated with the light ~rom strobe I. Since -~luorescence is extremely weak, the duration of irradiation with the exciting light should be as long as possible. This will be approximately several tens of' microseconds in view o~ the fact that a longer period o~ time may result in significant shaking o~ the image.
To be more precise, the timing for irradiation with the exciting light also must fall within the even-numbered field periods of the video camera 24. Accordin~ly, the timing at which the strobe light can be emitted when passage o~ a cell through the image capturing area has been moni-: tored falls within even-numbered fields up to 2.5 msec prior to the odd-numbe.r fieIds.
In operation, ~luorescence emitted by a cell in response to irradiation with the exciting light passes through the objec~ive lens 8 and is reflected b~ the beam-splitter 11, which has a high reflectance. The e~Yciting 7 ~ 2 light which has passed through the image capturing area is intercepted by an e~citing light-beam stopper 30, and stray light is removed by the filter 20. Near infrared light continuously emitted in order to monitor cell flow-through also is eliminated by the filter 20.
The fluorescent light which has passed through the filter 20 enters to the projecting lens 21, whereby an image of the cell is ~ormed on the photoelectric sur~ace of the image intensifier 22. At this time a high-voltage is applied to the image intensifier 22 so that the image is intensified by an internal MCP (a microchannel plate) to ~orm an image on the fluorescent output surface of the intensifier. This image, hal~ of which is masked by a semicircular mask 23, is re~Iected by a mirror 17 so as to pass through a relay lens 18, whereby an image is -formed on only hal~ o~ the CCD area sensor of the video camera 24 through a hal~-mirror 19.
Meanwhlle, the part of the ~luorescent light which has passed through the beam-splitter 11 is re~lected by the dichroic mirror 12 so that an image is ~ormed at the position of the semicircular mask 16. This image, however, is blocked by the mask. The part o~ the near in~rared light which has passed through the beam splitter 11 is almost totally transmitted by the dichroic mirror 12, and any part thereo~: reflected by the dichroic mirror 12 is eliminated .. by the ~ilter 20A. As a consequence, multiple exposure will not take place on the natural-light capturing side of the CCD area sensor of video camera 24 (already irradiated at ~ emission of the strobe light).
After a cell passing through the image capturing area is detected through a sequence of the aboYe kind, the image by white light~and ~luorescent image of the cell can be captured by the sole video camera 24. Fig. 4 illustrates an example of such an imaged ~rame. Fig. 3 is an e~ample illustrating the timing o~ strobe emission and excitation light emission after detection of a cell passin~
through the image capturing area, as well as the timing o-f gating signals for the image in-tensi~ier 22. The signals 9 ~ 2 for controlling such timing are produced by the discriminator/controller 28 shown in Fig. 1.
It is required that a cell be irradiated with the excitation light exactly when it has moved to the excitation-light irradiating area after passing through the image capturin~ area o~ the line sensor 14. It will suf~ice i~ control ~or such timing entails mere application o~ a fi~ed time delay ~ollowing detection o~ cell ~low-through, provided the ~low velocity o~ the cell does not ~luctuate. I~ ~low velocity fluctuates, however, the ~ollowing expedient can be adopted. Specifically, the position at which the ~luorescent i~age o~ the cell appears in one ~ra~e can readily be determined by image processin~.
There~ore, i~ this position shifts from the expected position ~rom one ~rame to the next, feedback control is applied so as to correct the time delay ~hich elapses until irradiation with the fluorescent light i9 per~ormed.
In the embodiment described above, the near in~rared semiconductor laser 45 is used as the light source ~or monitoring passage o~ cells through the-image capturing area. However, use can be made o~ a near in~rared LE~
instead. In addition, the positions at which the ~luo-rescent image light-detecting s~stem and white-light detecting system are disposed in Fig. 1 can be interchanged ~5 i~ desired. Furthermore, a~ arrangement can be adopted in which, by bringing the ~luorescent-light irradiating area to the upper side o~ the image capturing area in the same manner as the near infrared-light irradiating area, first the ~luorescent image is captured a~ter detection o~ cell ~low-through, and then the cell is irradiated with the ~strobe light a~ter waiting ~or the cell to move to the lower side o~ the image capturing area, whereby the image by white light is captured next.
The invention as described above a~ords the ~ollowing advantages:
(1) Since passage o~ cells through the image capturing area is monitored all time~, even the images of 2~7~2 cells in a weak concen-tration can be obtained efficiently and ~i~h e.Ycellen~ selectivity.
(2) The irradiating light for obtaining the fluorescent image of a cell need not irradiate the entire image capturing area o~ the video camera; it can be focused to a specific area instead. This makes it possible to raise the intensity of the irradiating light per unit area so that weak fluorescence can be cap~ured as an image even if e~posure time is short.
(3) Two images, namely the image by white light and the fluorescent image, can be acquired in one and the same imaged frame by a single video camera. This facilitates image analytical processing and has advantages in terms o~ cost.
--I
FLOW IMAGING CYTOMETER
BACKGROUND OF THE INVENTION:
1. Field of the Invention _ This invention relates to a ~low imaging cytometer.
More particularly, the invention relates to a flow-ima~in~
particle analyzing system in which cells fluorescently stained in a manner suitable for the particular cells o~
interest are introduced to a ~low cell to be ~ormed into a planar sheathed flow and irradiated with white light ~strobe light) to obtain an image by white light and e~cited with l~ser light to obtain a ~luorescent image in a highly efficient manner, and in which the two types of images can be captured simultaneously by a sin~le video camera and subject to analysis.
2. Description of the Prior Art -Attempts have been made to irradiate cells, wh:ich have been stained and smeared on a glass slide, with :Light such as risible light or ultraviolet light under a micro-scope, capture a ~luorescent image o~ cells o~ interest, analyze the resulting image and obtain physiolo~ical in~ormation relating -to the cells. However, a method o~
this ~ind is not suited to the analytical processing of a la~ge number of ceIls in a short time, and analysis using fluorescen~ lmages has only limited application.
In another example of the conventionaI flow cytometer, the cell information is obtained using a gross value o~ the fluorescence emitted from the fluorescently stained cell. In other words, the fluorescence emitted ~rom each portion o~ the cell is integrated over the entirety of the cell, and the cell information is obtained in the form of such an integrated value. Though such a method Iends i~sel~ ~o analysis of a large number of cells in a short period of time, it is not possi~le to acquire detailed information as to which portions o~ individual cells have been stained and caused to emit ~luorescence.
Consequently, this method is limited in terms of analytical performance.
2~7~
_ 7 _ On the other hand, a cell classi~ying apparatus ~hat has been put into practical use employs a technique in which cells stained in a manner suitable for a particular cell of interest are i.ntroduced to a ~low cell to be formed into a planar sheathed ~low and irradia-ted with strobe light, a still picture is ob-tained b~ a ~ideo camera and image processing is applied. However, the state of the art is such that the capturing and analysis of fluorescent images o~ individual cells using this method have still not reached a practical sta~e because o~ problems related to fluorescent i.maging sensitivity. The present invention makes use of the technology employed in a flow imaging cytometer of the type having a high image capturin~
efficiency, as previously proposed in the specification of Japanese Patent Application ~o. 185794/199O.
SUMMARY OF THE INVENTION:
Thus, the art still lacks a definitive ~low-imaging particle analyzing system Por sensing cells that pass through an image capturing area.and irradiating the cells with concentrated exciting light,. thereby to assure the required fluorescent intensity and obtain a fluorescent image, a~d for subjecting the fluorescent image, as well as a~ image by white light of the cells derived from the conventional white-light source, to hi~hly ef~icient image capturing and analysis using a single video camera.
Accordingly, an object of the present invention is to pro~ide a ~low imaging cytometer which e~pands upon the idea of the previously proposed (the aforementioned Japanese Patent Application No. 185794/1990. hereinafter referred to as "the earlier application'i) flow imaging cytometer of the type having a high image capturing efficiency, wherein fluorescence emitted by a fluorescently stained cell is obtained as a two-dimensional image at the same time as an image.by white light acquired by conventional strobe-light (white-light) irradiation.
According to the present invention, the foregoing object is attained by providing a flow imaging cytometer comprising a flow cell formed to include a flat flow -3- ~ 7~2 path f'or causing a specimen solution containing p~rticle components to be sensed ~o flow as a ~lat stream, ~irs~
and third light sources arranged on a ~irst side of the ~`low cell for irradiating the specimen solution in the flow cell with pulsed light, ~irst image capturing means arranged on an opposite side of the flow cell for picking up still pictures of the particle components in the specimen solution irradiated by the ~irst and third light s;ources, a second light source arranged on the eirst side o~ the ~low cell for irradiating the specimen solution in the flow cell with light continuously, second image capturing means arranged on the opposite side of the flow cell for picking up an image of the specimen solution irradiated by the second light source, processing means for executing prescribed analysis based upon image data ~rom the ~i.rst and seco:nd image capturing means, and control means ~or detecting the particle components based upon thè image data ~rom the second image capturlng means, and on the basis o~ such detection, ~or causing the third light source to emit light ~irst, ~ollowed by the first li~ht source upon passage of a prescribed time, within an image capturing period o~ the first image capturing means, wherein the first light source is a light source ~or e~citing fluorescence, the third light source ls a light source ~or emitting white light, and the image resultlng from the ~irst light source and the image resulting ~rom the third light source are each captured in a di~erent area on a light-detecting surface o~ the ~irst image capturing means.
The flow imaging cytometer o~ the present invention is further characterized in that the first image capturing means has a two-dimensional image capturing area on the ~low o~ the specimen solu~ion, the second image capturing means has a linear ima~e capturing area on ~he ~low o-~ the specimen solution, the image capturing area o~ the second image capturing means is ~ormed so as to cross the flow o~
the specimen solution within the image capturing area o~
the first image capturing means, the image capturing area o~ the ~irst image capturing means is divided into a zone 2~762 which includes, and a zone which does not include, the image capturing area of the second image capturing ~eans, and an image in one of these zones resulting from irradiation by the third light source and an image in the o~her o~ these zones resulting ~rom irradiation by the ~irst light source are captured by the first image capturing means.
The flow imaging cytometer of the present invention is further characterized by having masking means for masking light irradiating the ~irst image capturlng means in such a manner that the two ima~es do not overlap each other on the light-detecting surface of the first image capturing means.
The flow imaging cytometer of the present invention is further characterized by having means for forming the irradiating light ~rom the -first llght source into an elliptical shape, and in that the light-detectin~ system o~ a fluorescent image is provided with an image intensifier operated only when the ~luorescent image is captured.
Other features and advantages of the present invention will be apparent from the ~ollowing description taken in conjunction with the accompanying drawi~gs.
BRIEF DESCRIPTION OF THE DRAWINGS:
Fig. 1 is a block diagram illustrating the construction of a ~low imaging cytometer according to the present invention;
Fig. 2 is an e~planatory view illustrating an example o~ a light-irradiating area and an image capturing area of a flow~cell;
Fig. 3 is a timing chart illustrating irradiation timing and the timing of a gating signal for an image intensifier;
Fig. 4 is a diagram showing an example of an imaged frame of a cell captured by a video camera; and Fig. 5 is a diagram showing e.~amples of semicircular and rectangular masks.
35 DETAILED DESCRIPTION OF THE PREFERR_ EMBODI~lENTS:
A preferred embodiment of a flow imaging cytometer according to the present invention will now be described with reference to the drawings. The flow imaging cytometer ' ' ' . ' :' ' .
. . ' ' :' . ~; - :
. .
2~7~2 ;, includes, in addition to the light source (a near in-~rared semiconductor laser) and line sensor for monitoring passage o~ cells in the earlier application, an excitation light source for obtainin~ a fluorescent image~ an image in~en-sifier for intensifying the fluorescence, and variousmirrors, filters and masks e~ployed so that the fluorescent image as well as a conventional image by white light can be acquired by a single video camera.
As shown in Fig. 1, the flow imaging cytometer o~
the invention includes a planar-sheath flow cell 6 to which a specimen solution containing stained cells is introduced.
In order that passage of these cells through an image cap-turing area of a video camera 24 may be monitored at all times, the image capturing area is irradiated continuously with laser light -~rom a near ln~rared semiconductor laser 45. The light ~rom the semiconductor laser 45 is colli-mated by a collimator lens 46, and the collimated light is reflected by a dichroic mirror 4 upon passing through a cylindrical lens 47. The reflected light is stopped down to a finely elongated beam spot perpendicular to the direction of cell move by a condenser lens 5 and irradiates an image capturing area o~ a line sensor 14, as illustrated in Fig. 2. In this embodiment, the image capturing area of the line sensor 14 is provided slightly above mid-center of the upper half of the image capturing area of video camera 24.
The light from the semiconductor laser 45 leaving the image capturing area passes through an objective lens 8 a~d is then split by a beam-splitter il. Part of the light ~rom the beam-splitter 11 passes through a dichroic mirro~
;12 and enters to a projecting lens 13, which proceeds to Yorm an imaFe on the line sensor 14. The line sensor 14 successively produces voltage outputs conforming to the accumulated amount of photoelectrical conversion of each pixel s~posed for a scannin~ cycle (several tens of microseconds) o~ one line. By means of signal processing similar to that set forth in -the earlier application, a trigger for strobe-light irradiat~on is applied when :
2 ~ 2 C~
a cell crosses the image cap~uring area of the line sensor 1~ during e~en-numbered field interYals of the ~ideo camera 24.
The processing time from the instant a cell crosses the image capturing area of the line sensor 14 until a strobe 1 is triggered is lOo - 200 ~sec î~ the scanning cycle of the line sensor 14 is 5~ ~sec. On the assumption that the ~low velocity of cells in flow cell 6 is 30 ~m/sec, a cell will move 3 - 6 ~m in this period o~ time. Accord-ingly, the image of a cell obtained by being irradiated withthe strobe 1 will always fall in the area located in the upper half o~ one imaged ~rame, as illustrated in Fig. 4.
The strobe light from strobe 1 is collimated by a collimator lens 9, the collimated light passes through a condenser lens 10 and dichroic mirrors 4, 4A and enters to the condenser lens 5, by virtue o~ which the entirety o~
the image capturing area of video camera 24 is irradlated with the strobe light substantially uniPormly. This strobe light which has passed through the image capturing area is reflected by the dichroic mirror 12 upon being acted upon by the objective lens 8 and beam-splitter 11. The reflected light has its near infrared component cut by a ~ilter 20A, with the resulting light entering to a projecting lens 15.
The latter forms an image upon a semicircular mask 16. shown in Fi~. 5. The lower hal~ of the image is blocked by the . mask 16, as a result of which an image is ~ormed on only hal~ o~ a CCD area sensor of the video camera 24 via a rela~
lens 27 and half-mirror 19.
The part of the light reflected by the beam-splitter 11 passes through a filter 20 and a projecting lens 21, whereby an image is formed on the photoelectric surface of image~intensifier 22. Since gating is applied in such a manner that a voltage is not impressed upon the image intensifier 22 at this poin~ in time, an image does not appear on its output sur~ace. A gating signal ~or this purpose is produced by a discriminator/controller 28, which i5 ~or judging when a cell has passed through the ima~e capturing area, and for controlling the light sources.
.
.
7 ~ 2 ~ ter a cell has been irradiated with light from the strobe 1, the system wai.ts for the cell to travel to a position in the lower half of the image ca~turing area before irradiating the cell with light from an e~citation light source 35 (for example, an He-Cd laser or xenon lamp).
A trigger signal for this purpose is produced by the light-source controller 28. The light from the light source 35 is rendered into an oblong form by a cylindrical lens 36, and the light ~rom lens 36 is re~lected by the dichroic mirror 4A. The reflected light is stopped down to a ~inely elongated beam spot perpendicular to the direction of cell move by the condenser lens 5 and irradiates the mid-center region o~ the lower half of the image capturing area o~
video camera 24t as depicted in Fig. 2. The cell will be moving through this irradiated area at this time. If the moving speed of the cell through the flow cell 6 is 30 mm~sec and the image capturing area o~ the video camera 24 has a size of 150 ~ 150 ~m, then control should be exer-cised in such a manner that the image capturing area is irradlated with the exciting light approximately 2.5 msec a~ter this area has been irradiated with the light ~rom strobe I. Since -~luorescence is extremely weak, the duration of irradiation with the exciting light should be as long as possible. This will be approximately several tens of' microseconds in view o~ the fact that a longer period o~ time may result in significant shaking o~ the image.
To be more precise, the timing for irradiation with the exciting light also must fall within the even-numbered field periods of the video camera 24. Accordin~ly, the timing at which the strobe light can be emitted when passage o~ a cell through the image capturing area has been moni-: tored falls within even-numbered fields up to 2.5 msec prior to the odd-numbe.r fieIds.
In operation, ~luorescence emitted by a cell in response to irradiation with the exciting light passes through the objec~ive lens 8 and is reflected b~ the beam-splitter 11, which has a high reflectance. The e~Yciting 7 ~ 2 light which has passed through the image capturing area is intercepted by an e~citing light-beam stopper 30, and stray light is removed by the filter 20. Near infrared light continuously emitted in order to monitor cell flow-through also is eliminated by the filter 20.
The fluorescent light which has passed through the filter 20 enters to the projecting lens 21, whereby an image of the cell is ~ormed on the photoelectric sur~ace of the image intensifier 22. At this time a high-voltage is applied to the image intensifier 22 so that the image is intensified by an internal MCP (a microchannel plate) to ~orm an image on the fluorescent output surface of the intensifier. This image, hal~ of which is masked by a semicircular mask 23, is re~Iected by a mirror 17 so as to pass through a relay lens 18, whereby an image is -formed on only hal~ o~ the CCD area sensor of the video camera 24 through a hal~-mirror 19.
Meanwhlle, the part of the ~luorescent light which has passed through the beam-splitter 11 is re~lected by the dichroic mirror 12 so that an image is ~ormed at the position of the semicircular mask 16. This image, however, is blocked by the mask. The part o~ the near in~rared light which has passed through the beam splitter 11 is almost totally transmitted by the dichroic mirror 12, and any part thereo~: reflected by the dichroic mirror 12 is eliminated .. by the ~ilter 20A. As a consequence, multiple exposure will not take place on the natural-light capturing side of the CCD area sensor of video camera 24 (already irradiated at ~ emission of the strobe light).
After a cell passing through the image capturing area is detected through a sequence of the aboYe kind, the image by white light~and ~luorescent image of the cell can be captured by the sole video camera 24. Fig. 4 illustrates an example of such an imaged ~rame. Fig. 3 is an e~ample illustrating the timing o~ strobe emission and excitation light emission after detection of a cell passin~
through the image capturing area, as well as the timing o-f gating signals for the image in-tensi~ier 22. The signals 9 ~ 2 for controlling such timing are produced by the discriminator/controller 28 shown in Fig. 1.
It is required that a cell be irradiated with the excitation light exactly when it has moved to the excitation-light irradiating area after passing through the image capturin~ area o~ the line sensor 14. It will suf~ice i~ control ~or such timing entails mere application o~ a fi~ed time delay ~ollowing detection o~ cell ~low-through, provided the ~low velocity o~ the cell does not ~luctuate. I~ ~low velocity fluctuates, however, the ~ollowing expedient can be adopted. Specifically, the position at which the ~luorescent i~age o~ the cell appears in one ~ra~e can readily be determined by image processin~.
There~ore, i~ this position shifts from the expected position ~rom one ~rame to the next, feedback control is applied so as to correct the time delay ~hich elapses until irradiation with the fluorescent light i9 per~ormed.
In the embodiment described above, the near in~rared semiconductor laser 45 is used as the light source ~or monitoring passage o~ cells through the-image capturing area. However, use can be made o~ a near in~rared LE~
instead. In addition, the positions at which the ~luo-rescent image light-detecting s~stem and white-light detecting system are disposed in Fig. 1 can be interchanged ~5 i~ desired. Furthermore, a~ arrangement can be adopted in which, by bringing the ~luorescent-light irradiating area to the upper side o~ the image capturing area in the same manner as the near infrared-light irradiating area, first the ~luorescent image is captured a~ter detection o~ cell ~low-through, and then the cell is irradiated with the ~strobe light a~ter waiting ~or the cell to move to the lower side o~ the image capturing area, whereby the image by white light is captured next.
The invention as described above a~ords the ~ollowing advantages:
(1) Since passage o~ cells through the image capturing area is monitored all time~, even the images of 2~7~2 cells in a weak concen-tration can be obtained efficiently and ~i~h e.Ycellen~ selectivity.
(2) The irradiating light for obtaining the fluorescent image of a cell need not irradiate the entire image capturing area o~ the video camera; it can be focused to a specific area instead. This makes it possible to raise the intensity of the irradiating light per unit area so that weak fluorescence can be cap~ured as an image even if e~posure time is short.
(3) Two images, namely the image by white light and the fluorescent image, can be acquired in one and the same imaged frame by a single video camera. This facilitates image analytical processing and has advantages in terms o~ cost.
(4) Images of a large number of cells per unit time can be obtained and subJected to analytical processing by ~low imaging techniques.
As many apparently widely di~ferent embodiments o~
the present invention can be made without departing from the spirit and scope thereof, it is to be understood that the invention is not limited to the speci~ic embodime~ts thereof except as defined in the appended claims.
As many apparently widely di~ferent embodiments o~
the present invention can be made without departing from the spirit and scope thereof, it is to be understood that the invention is not limited to the speci~ic embodime~ts thereof except as defined in the appended claims.
Claims (5)
1. A flow imaging cytometer comprising:
a flow cell formed to include a flat flow path for causing a specimen solution containing particle components to be sensed to flow as a flat stream;
first and third light sources arranged on a first side of said flow cell for irradiating the specimen solution in said flow cell with pulsed light;
first image capturing means arranged on an opposite side of said flow cell for capturing still pictures of the particle components in the specimen solution irradiated by said first and third light sources;
a second light source arranged on the first side of said flow cell for irradiating the specimen solution in said flow cell with light continuously;
second image capturing means arranged on the opposite side of said flow call for picking up an image of the specimen solution irradiated by said second light source;
processing means for executing prescribed analysis based upon image data from said first and second image capturing means; and light-source control means for detecting the parti-cle components based upon the image data from said second image capturing means, and on the basis of such detection, for causing said third light source to emit light first, followed by said first light source upon passage of a prescribed time, within an image capturing period of said first image capturing means;
wherein said first light source is a light source for exciting fluorescence, said third light source is a light source for emitting white light, and the image resulting from said first light source and the image resulting from said third light source are each captured in a different area on a light-detecting surface of said first image capturing means.
a flow cell formed to include a flat flow path for causing a specimen solution containing particle components to be sensed to flow as a flat stream;
first and third light sources arranged on a first side of said flow cell for irradiating the specimen solution in said flow cell with pulsed light;
first image capturing means arranged on an opposite side of said flow cell for capturing still pictures of the particle components in the specimen solution irradiated by said first and third light sources;
a second light source arranged on the first side of said flow cell for irradiating the specimen solution in said flow cell with light continuously;
second image capturing means arranged on the opposite side of said flow call for picking up an image of the specimen solution irradiated by said second light source;
processing means for executing prescribed analysis based upon image data from said first and second image capturing means; and light-source control means for detecting the parti-cle components based upon the image data from said second image capturing means, and on the basis of such detection, for causing said third light source to emit light first, followed by said first light source upon passage of a prescribed time, within an image capturing period of said first image capturing means;
wherein said first light source is a light source for exciting fluorescence, said third light source is a light source for emitting white light, and the image resulting from said first light source and the image resulting from said third light source are each captured in a different area on a light-detecting surface of said first image capturing means.
2. The flow imaging cytometer according to claim 1, wherein said first image capturing means has a two-dimensional image capturing area on the flow of the specimen solution, said second image capturing means has a linear image capturing area on the flow of the specimen solution, the image capturing area of said second image capturing means is formed so as to cross the flow of the specimen solution within the image capturing area of said first image capturing means, the image capturing area of said first image capturing means is divided into a zone which includes, and a zone which does not include, the image capturing area of the second image capturing means, and an image in one of these zones resulting from irradiation by said third light source and an image in the other of these zones resulting from irradiation by said first light source are captured by said first image capturing means.
3. The flow imaging cytometer according to claim 2, further comprising masking means for masking light irra-diating said first image capturing means in such a manner that the two images do not overlap each other on the light-detecting surface of said first image capturing means.
4. The flow imaging cytometer according to claim 2, further comprising means for forming the irradiating light from said first light source into an elongated elliptical shape.
5. The flow imaging cytometer according to any one of claims 1 through 4, wherein a light-detecting system of a fluorescent image is provided with an image intensifier, and said image intensifier is operated only when the fluorescent image is captured.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33137/1991 | 1991-02-27 | ||
| JP3033137A JPH0734012B2 (en) | 1991-02-27 | 1991-02-27 | Flow image cytometer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2050762A1 true CA2050762A1 (en) | 1992-08-28 |
Family
ID=12378212
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002050762A Abandoned CA2050762A1 (en) | 1991-02-27 | 1991-09-05 | Flow imaging cytometer |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US5159397A (en) |
| EP (1) | EP0501008B1 (en) |
| JP (1) | JPH0734012B2 (en) |
| CA (1) | CA2050762A1 (en) |
| DE (1) | DE69126120T2 (en) |
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| WO2022268325A1 (en) | 2021-06-24 | 2022-12-29 | Leica Microsystems Cms Gmbh | Calibration object for calibrating an imaging system |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3992112A (en) * | 1975-09-29 | 1976-11-16 | Corning Glass Works | Attenuating image extender for multiple imaging system |
| US4338024A (en) * | 1980-05-02 | 1982-07-06 | International Remote Imaging Systems, Inc. | Flow analyzer and system for analysis of fluids with particles |
| US4573796A (en) * | 1984-01-06 | 1986-03-04 | The United States Of America As Represented By The United States Department Of Energy | Apparatus for eliminating background interference in fluorescence measurements |
| JPS60260830A (en) * | 1984-06-07 | 1985-12-24 | Fujisawa Pharmaceut Co Ltd | Light source device for irradiating cell in automatic analysis instrument for cell |
| US4786165A (en) * | 1986-07-10 | 1988-11-22 | Toa Medical Electronics Co., Ltd. | Flow cytometry and apparatus therefor |
| JPH073419B2 (en) * | 1986-10-07 | 1995-01-18 | 東亜医用電子株式会社 | Method and device for analyzing cells in fluid |
| WO1990009637A1 (en) * | 1989-02-13 | 1990-08-23 | Research Corporation Technologies, Inc. | Method and means for parallel frequency acquisition in frequency domain fluorometry |
| US5093866A (en) * | 1990-02-09 | 1992-03-03 | Hamilton Equine Associates Limited | Fluorescence and motility characterization system for cells, bacteria, and particles in fluids |
-
1991
- 1991-02-27 JP JP3033137A patent/JPH0734012B2/en not_active Expired - Lifetime
- 1991-09-05 US US07/755,304 patent/US5159397A/en not_active Expired - Lifetime
- 1991-09-05 EP EP91115007A patent/EP0501008B1/en not_active Expired - Lifetime
- 1991-09-05 DE DE69126120T patent/DE69126120T2/en not_active Expired - Fee Related
- 1991-09-05 CA CA002050762A patent/CA2050762A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| DE69126120D1 (en) | 1997-06-19 |
| JPH04270961A (en) | 1992-09-28 |
| EP0501008B1 (en) | 1997-05-14 |
| US5159397A (en) | 1992-10-27 |
| EP0501008A2 (en) | 1992-09-02 |
| DE69126120T2 (en) | 1997-10-09 |
| JPH0734012B2 (en) | 1995-04-12 |
| EP0501008A3 (en) | 1992-11-04 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |