WO2001084109A1

WO2001084109A1 - Analysis plate and method of making and using same

Info

Publication number: WO2001084109A1
Application number: PCT/US2001/014210
Authority: WO
Inventors: Zonh-Zen Ho; Robert A. Lieberman; Gajendra D. Savant; Tomasz P. Jannson; Allan Wang
Original assignee: Physical Optics Corporation
Priority date: 2000-05-04
Filing date: 2001-04-30
Publication date: 2001-11-08
Also published as: AU2001257497A1; TW491941B

Abstract

Analysis of chemical samples is performed using a substrate having a three-dimensional light shaping sample surface (108). The three-dimensional light shaping sample surface promotes sample adhesion and optical analysis of the samples. The substrate (106) is a substantially optically transparent material having formed in one surface thereof the three-dimensional light shaping structure. Samples such as chemicals may be placed on the surface of the substrate comprising the three-dimensional light shaping structure. The light shaping structures are arranged to enhance and increase the available surface area of the substrate to the sample as well as optical analysis of the sample while on the substrate. The substrate may be used in many forms such as a monolith, a microtiter plate (100), a chamber, a flask, and others.

Description

ANALYSIS PLATE AND METHOD OF MAKING AND USING SAME

Background of the Invention

1. Field of the Invention

The present invention relates generally to analyzing chemical samples, and more particularly, to an analysis substrate having a three-dimensional light shaping sample surface and a method of making such a substrate.

2. Discussion of the Related Art

Substrates are widely used for a variety of chemical testings or assays. Using these tests, chemicals can be accurately quantified and characterized. These tests are used in laboratory research and clinical diagnoses of many common diseases, such as cancer, inflammatory diseases, AIDS, hepatitis, and immunological diseases. Approximately fifty million plates are used every year in the United States. In 1996, the market for various plates was approximately eighty million dollars. However, the conventional flat plate has a small surface area for samples to attach. While current plasticware is available in a variety of shapes and sizes, this plasticware has flat two-dimensional surfaces with limited surface area. Several attempts have been made to increase the surface area of plasticware. Additionally, grooved well bottoms, which increases the surface area of the well, can be constructed. Currently, laboratory plasticware exists in many shapes including flasks,

Petri dishes, multi-well plates (including microtiter plates), roller bottles, and tubes. Tissue culture dishes come in 35, 60, 90, 100, and 145 diameters, with a variety of modifications (e.g., internal wells and grids). Generally, this plasticware has lids that are well vented for adequate gas exchange, but minimum evaporation. Multi- well plates can have several different shaped bottoms, including flat, round, V, and U bottoms.

Additionally, today microtiter plates are being produced with an increasing number of wells. For example, one manufacturer offers a plate with 4,356 wells per cubic centimeter for high-density cell or molecular assays. This tremendous increase in the density of wells produces a concomitant decrease in well size and surface area. This decrease in well size and surface area produces a need for increasing a well's surface area. For apparatus used with analytical instrumentation, such as high-pressure liquid chromatography (HPLC) and gas liquid chromatography (GLC), where chemical sensing is based on the substrate coming into contact with a substance, the substrates limited such analysis by the limited surface area of the planar surfaces, and also smooth spherical surfaces or substrates used with such analytical instruments.

For chemical assays what is needed is a substrate with greater surface areas. Additionally, there was a need for a substrate that could be engineered to enhance the surface area of substrates.

Additionally, analysis apparatus and samples may be illuminated for purposes of analysis using adsorbance, fluorescence and luminescence analysis techniques. In the prior art, both fluorescence and luminescence using incident light photometry are top counting analysis techniques. Transmitted illumination, i.e., clear bottom plates, allow bottom detector arrangements. However, prior art clear bottom plates offer no optical shaping of the light emanating from the sample and do not reduce backscatter of source light from the plate. Reflection, refraction, scattering, misdirection of light, and other optical losses result in only a small percentage of transmitted illumination reaching the detector, degrading both read accuracy and read time. Also, with increasing density of wells in a microtiter plate there is significantly less illuminated area per well. Thus, what is also needed is analysis apparatus having optical shaping of light emanating from the sample, which reduces backscatter of source light from the plate.

Summary of the Invention An object of the invention is to provide three-dimensional light shaping structures to enhance chemical sample analysis. The light shaping structures promote adhesion and analysis of the samples. In accordance with a first aspect of the invention, this object is achieved by using an analysis apparatus comprising a substantially optically transparent material, the apparatus having top and bottom surfaces and the top surface formed to include a three-dimensional light shaping structure.

Another object of the invention is to provide an apparatus and method for concentrating chemicals. In accordance with this aspect of the invention, the chemicals are concentrated on an apparatus that has a three-dimensional light shaping structure, which increases surface area beneficial to chemical concentration. Still another object of the invention is to provide a microtiter plate comprising a substantially optically transparent substrate, the substrate having first and second surfaces and a plurality of analysis wells in the first surface, each well having a sidewall and a bottom defining an analysis chamber and wherein the bottom surface is formed to include a three-dimensional light shaping structure. Yet another object of the invention is to provide a microtiter comprising a substantially optically transparent monolith, the monolith having top and bottom surfaces and formed to include three-dimensional light shaping structures and a well array having first and second sides and a plurality of passages formed therein, each passage having first and second open ends and a sidewall and wherein the well array is secured to the top surface such that each passage defines a fluid tight analysis chamber defined by the sidewall and the top surface.

Another object of the invention is to provide an analysis flask comprising a monolith of substantially optically transparent material, the monolith having top and bottom surfaces, the top surface formed to include a three-dimensional light shaping structure, and an analysis chamber secured to the monolith and over the top surface and defining a fluid tight chamber.

It is another object of the invention to provide a method of analyzing samples comprising the steps of providing a substantially optically transparent substrate, the substrate having first and second surfaces and placing the sample into at least one of a plurality of analysis wells formed in the first surface, each well having a sidewall and a bottom defining an analysis chamber and wherein the bottom is formed to include a three-dimensional light shaping structure, and adhering the sample to the three-dimensional light shaping structure.

It is still another object of the invention to provide an apparatus for chemical sample analysis which reduces backscatter of light from a light source during optical analysis of the sample and thereby enhances detection of the desired light signal emanating from the sample and thereby increases the signal to noise ratio during analysis.

It is still another object of the invention to provide an apparatus for chemical sample analysis which shapes the light emanating from the sample into an appropriate light output distribution so that less of the light emanating from the sample is wasted and more is available to be detected and analyzed.

In accordance with a still further object of the invention, the substrate and the sample in the analysis wells are illuminated with light from a light source, and the light shaping structures shape the light emanating from the sample into a desired light output distribution which is then detected and analyzed.

It is yet another object of the invention to provide a method of analyzing a sample on an analysis substrate comprising the steps of providing a substantially optically transparent substrate having a surface formed to include a three- dimensional light shaping structure, placing the sample onto the substrate and the three-dimensional light shaping structure, providing a source of light, illuminating the substrate and the sample with the light, the light shaping structure shaping the light emanating from the sample into a desired light output distribution and reducing backscatter of light incident the substrate and sample, and detecting and analyzing the light output distribution. Other objects, features, and advantages of the invention will become apparent to those skilled in the art from the following detailed description and the accompanying drawings. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the present invention without departing from the spirit thereof, and the invention includes all such modifications.

Brief Description of the Drawings Preferred exemplary embodiments of the invention are illustrated in the accompanying drawings in which like reference numerals represent like parts throughout, and in which:

Figure 1 is a perspective view of a microtiter plate arranged in accordance with a preferred embodiment of the present invention; Figure 2 is a partial cross-section view taken along line 2-2 of Figure 1;

Figure 3 is a plan view of microtiter plate having analysis wells arranged in accordance with a first alternate preferred embodiment of the present invention;

Figure 4 is a plan view of a microtiter plate having analysis wells arranged in accordance with a second alternate preferred embodiment of the present invention; Figure 5 is a partial cross-section view of a microtiter plate having analysis wells arranged in accordance with a third alternate preferred embodiment of the present invention;

Figure 6 is a perspective view of an analysis flask arranged in accordance with a preferred embodiment of the present invention; Figure 7 is a cross-section view taken along line 7-7 of Figure 6;

Figure 8 is a schematic illustration of an analysis system adapted for use with the preferred embodiments of the present invention;

Figure 9 is a schematic representation of a light distribution pattern in accordance with a preferred embodiment of the present invention; Figure 10 is a photograph of three-dimensional light shaping surface structure arranged to produce the light distribution pattern illustrated in Figure 9;

Figure 11 is a schematic representation of a light distribution pattern in accordance with an alternate preferred embodiment of the present invention; Figure 12 is a photograph of a three-dimensional light shaping surface structure arranged to produce the light distribution pattern illustrated in Figure 11 ; Figure 13 is a schematic representation of an apparatus for forming a microtiter plate in accordance with a preferred embodiment of the present invention;

Figure 14A is a schematic representation of the dimensional characteristics of a three-dimensional structure in accordance with a preferred embodiment of the present invention;

Figure 14B is a schematic representation of the dimensional characteristics of a three-dimensional structure in accordance with an alternate preferred embodiment of the present invention; and

Figure 15 is a partial cross-section view illustrating an analysis plate in accordance with an alternate preferred embodiment of the present invention.

Detailed Description of the Preferred Embodiments 1. Analysis Apparatus Incorporating Three-dimensional Structures A. Microtiter Plate In view of the foregoing, and with reference to Figures 1 and 2, a microtiter plate 100 includes a plurality of analysis wells 102. Each analysis well 102 has a generally cylindrical sidewall 104 extending above a substantially planar substrate 106 defining a bottom 108. Microtiter plate 100 is preferably formed from a plastic material such as polystyrene, polycarbonate, polyvinylchloride (PNC) or the like chosen for its optical transmission properties. Microtiter plate 100 may be formed to include a number of analysis wells 102 with six such analysis wells being shown in Figure 1 for purposes of illustrating the present invention.

While each analysis well 102 is shown to have a cylindrical shape, it will be appreciated that other shapes may be used without departing from the fair scope of the present invention. For example, and with reference to Figures 3-5 several alternative arrangements for analysis wells, respectively labeled 102' , 102" , and 102"' , are shown. As illustrated in Figure 3, analysis wells 102' have a round cylinder shape. Alternatively, each analysis well 102" may have a prismatic, i.e. , rectangular shape, to enhance well density. Equally suitable is an analysis well 102" , as shown in Figure 4, having a prismatic shape with corners rounded to a radius "r." Of, course for further increased well density prismatic shaped wells with square corners may be used. Figure 5 illustrates an analysis well 102"' not having a right cylinder configuration. That is, bottom 108'" does not form a right angle to sidewall 104"' . Instead, analysis well 102"' has a generally "tulip" or parabolic shaped bottom 108'" . It will be appreciated that numerous additional shapes including "U" bottom, "V" bottom and others may be utilized in order to enhance well density, increase surface area, increase volume, and the like as required for a particular analysis application. Referring once again to Figure 2, each bottom 108 is formed to include a three-dimensional structure arranged to enhance the optical properties of microtiter plate 100.

B. Fabricating the Three-dimensional Structures It has been found that a three-dimensional structure utilized in diffuser technology, such as the diffuser structures disclosed in commonly assigned United States Patent Nos. 5,365,354; 5,534,386 and 5,609,939 the disclosures of which are hereby expressly incorporated herein by reference, provide an unexpectedly sample friendly environment. As described therein, a three-dimensional light shaping structure comprising random, disordered, and non-planar speckle may be recorded and developed in a photosensitive medium so that the medium has non-discontinuous and smoothly varying changes in its refractive index (e.g., a gradient refractive index) which scatter collimated light into a controlled pattern with smooth brightness variation. A three-dimensional surface structure formed in this manner may be replicated in any number of materials, transparent or nontransparent, and then be adapted to form bottom 108.

More particularly, a coherent laser light source coupled through an objective lens may be used to record the three-dimensional surface structure from a master diffuser into a photosensitive medium. The master diffuser may be a standard ground glass diffuser, a lenticular diffuser, an acetate diffuser or a holographic diffuser. The size, shape, and orientation of the surface features recorded in the photosensitive medium is a function of a number of variables including the type of objective lens and master diffuser used, as well as the relative positioning of those components with respect to each other and with respect to the photosensitive medium. Ultimately, the desired results are obtained through empirical testing, and particularly, a three-dimensional structure most suitable for adhering and/or culturing a particular sample will be arrived at through such testing.

A generalized statement as to the effect of the formation parameters on the three-dimensional surface structure may be made and will assist in selecting an arrangement for achieving a desired three-dimensional surface structure. The objective lens expands the coherent light so that the area of incidence of light from the objective lens on the master diffuser is larger than that of the cross section of the laser beam itself. Small magnification by the objective lens results in a smaller incidence on the master diffuser. In this case, the features recorded on the photosensitive medium are larger. Thus, the size of the aperture of light incident the master diffuser is inversely related to the size of the surface features recorded in the photosensitive medium.

The distance between the objective lens and the master diffuser also affects the size of the surface structure. As the distance between the objective lens and the master diffuser decreases, the size of the surface structure increases. This occurs because as the objective lens moves closer to the master diffuser the apparent aperture of light incident the master diffuser is smaller. Increased structure size provides a three-dimensional surface structure with reduced optical diffusion, i.e., a smaller angular light distribution. Conversely, if the distance between the objective lens and the master diffuser increases, the size of the surface structure decreases. Smaller surface structure results in a larger angular spread.

The distance between the master diffuser and the photosensitive medium also affects speckle size. As the distance decreases, the size of the speckle recorded in the photosensitive medium decreases as well. This occurs because, assuming an expanded beam of light is produced at the objective lens, as the photosensitive medium is moved closer to the master diffuser, the lightbeam emanating from each of the irregularities in the master diffuser will expand less by the time it reaches the photosensitive medium, thus producing smaller speckle. Conversely, if the distance between the master diffuser and the photosensitive medium is increased, the size of the speckle recorded will be increased. Thus, these simple relationships between the object lens, the master diffuser, and the photosensitive medium are all adjusted, empirically, to achieve the size of the speckle desired in the photosensitive medium which in turn determines the light output distribution of a diffuser made from the photosensitive medium as further described below. The photosensitive medium may also be recorded using other methods including incoherent light sources and contact printing processes as is fully described in commonly assigned, copending U.S. Application Serial No. 09/137,397, filed August 20, 1998 (in which a CPA application was filed on February 29, 2000), a continuing patent application of U.S. Application No. 09/075,023, filed May 8, 1998 (now abandoned), the disclosure of which is hereby expressly incorporated herein by reference.

Most preferably a three-dimensional surface structure having the desired light distribution pattern is created using the above described processes and is used to create a durable metal master having the desired three-dimensional surface structure embossed therein using standard plating techniques. More particularly, the three-dimensional surface structure is recorded in the photosensitive medium, which is then developed. From the photosensitive medium a replica structure, preferably made by curing a layer of epoxy over the three-dimensional surface structure in the photosensitive material, is made. The cured epoxy may be removed from the photosensitive material leaving the three-dimensional surface structure recorded therein.

A thin layer of silver metal is then deposited and metalized, onto the cured epoxy. A cathode is then coupled to the silver, and an anode is coupled to a nickel bar. The silver coated epoxy and the nickel bar are then submerged in an acid bath, and a current is passed through the silver, the bath, and the nickel bar, causing a layer of nickel to form on the silver. The layer may develop to be up to 0.5 inch thick. The epoxy, silver, and nickel structure is then removed from the bath and the epoxy and silver are removed from the nickel leaving a metal master, i.e., a nickel shim with the three-dimensional surface structure formed therein. With reference to Figure 13, a microtiter mold 200 incorporating a metal master 208 formed in the manner described above is shown. Mold 200 includes a lower mold die 204 and an upper mold die 206. The metal master 208 is soldered, braised, welded, bonded or otherwise suitably secured to the lower mold die 204 taking care not to distort the three-dimensional surface structure formed therein. A mold cavity 210 is then formed in lower mold die 204 such as by electric discharge machining (EDM) milling or the like. The mold cavity defines a complement to the shape of a microtiter plate. As is known in the art, upper mold die 206 encloses mold cavity 210, and plastic material is injected into mold cavity from material source 202. It will be appreciated that mold cavity 210 is formed with suitable relief and draft angles to facilitate removal of a completed microtiter plate. In addition, various release compounds may also be used.

C. Monolith. Flask, and Concentrators Incorporating Three-dimensional Light Shaping Structures

The above method of manufacture may be used to form analysis apparatuses of numerous configurations including microtiter plate 100. The simplest such structure is a flat analysis plate, i.e., a monolith, having a surface formed to include a three-dimensional surface structure. With reference to Figure 6 and Figure 7, an analysis flask 300 constructed in accordance with preferred embodiments of the present invention is shown. Analysis flask 300 incorporates a sample plate 302. Plate 302 has a sample surface 304 and a bottom surface 306. Sample surface 304 is preferably formed to include a three-dimensional surface structure 308 as described herein. Enclosing sample surface 304 is an optically transparent housing 310 hermetically sealed about a periphery of plate 302. An aperture 312 is formed in housing 310 and a plug 314 is provided for permitting introduction and sealing of material within flask 300. Of course, where the housing 310 is used only for growing cells, it need not be transparent.

Referring to Figure 15, another analysis apparatus 400 is formed in a plate 402. More particularly, plate 402 is formed to include at least one passage 404 extending from a top surface 406 through plate 402 to a bottom surface 408. Passage 404 defines an interior wall 410. As shown, passage 404 has a tapered or "V" configuration. It will be appreciated that other configurations may be incorporated. Also, passage 404 may define an elongate slit in plate 402 or may define a cylindrical or frusto-conical aperture. Interior wall 410 is formed to include a three-dimensional surface structure 412. Sample material may be passed through passage 404 with a portion of the material being retained by adhesion within three- dimensional surface structure 412. In addition, a seal 414 may be provided to seal a bottom portion 416 of passage 404. In such an arrangement, passage 404 is configured as an analysis well with sample material retained therein. Seal 414 may subsequently be removed to allow sample material to drain from passage 404. Several steps of inoculating and draining or passing samples through passage 404 may thereby be accomplished. The accumulated adhered sample material in three- dimensional surface structure 412 may then be analyzed using one of several analysis techniques.

Referring to Figure 8, another exemplary analysis apparatus 500 is shown for analyzing samples prepared in a microtiter plate, such as microtiter plate 100. Apparatus 500 includes a light source 502, a detector 504, and a processor 506. Light source 502 is arranged to illuminate one or more analysis wells 102 of microtiter plate 100 with a coherent laser light beam. In accordance with the preferred embodiments of the present invention, a light distribution 508 from the illuminated analysis well is optically shaped by the three-dimensional surface structure formed in bottom 108. For microtiter plate 100 the light distribution is preferably a circular distribution having an angle ψ of about 1 - 10 degrees spread, and most preferably, for a CCD detector, about 2 - 3 degrees spread (see the distribution illustrated in Figure 9 and the corresponding structure illustrated in Figure 10). By incorporating the three-dimensional surface structure in bottom 108 substantially more of the incident light is directed towards detector 504, and less is wasted. Detector 504 is preferably a charge-coupled device (CCD) having an output coupled to processor 506 arranged to process and analyze the sample data.

Another important benefit of using the inventive microtiter plate or any other sample plate or chamber incorporating the three-dimensional surface structure of the present invention, is that light from source 502 incident the microtiter plate 100 is not back scattered toward the light source 502. Instead, it is transmitted through to the sample where it interacts with the sample. This increases the intensity of the light interacting with and emanating from the sample.

In any type of spectroscopic system, be it Raman or any other, light of the light source's wavelength that strays outside of the sample is undesirable, because it reduces the signal to noise ratio of the system, making it more difficult to distinguish between the desired light signal from the sample and light from the source. Particularly in Raman spectroscopy, the intensity of the light emanating from the sample at a wavelength slightly shifted from the wavelength of the light source is low compared to the intensity of the source light making it difficult to detect the desired signal apart from the source light. Low signal to noise ratios are therefore a problem in prior art systems for optical sample study. Because the inventive plates significantly reduce backscatter, the inventive plate can significantly improve the signal to noise ratios in most spectroscopic systems. Microtiter plates formed in accordance with the preferred embodiments are capable of transmitting more than 80 percent of the incident light to detector 504. In contrast, prior art microtiter plates, because of backscatter ing and uncontrolled diffusion, may transmit less than 10 percent of the incident light to detector 504. Moreover, the broad distribution of light from the prior art microtiter plates can result in cross talk at detector 504. That is, adjacent pixels of the CCD corresponding to other analysis wells of the microtiter plate may be illuminated resulting in false indications. With higher density microtiter plates it becomes increasingly more important to direct a higher percentage of the incident light towards the detector in a controlled pattern. As noted above, an advantage of forming the three-dimensional surface structure in this manner is that the light distribution pattern from the diffuser may be controlled. This is in contrast to conventional diffuser s that do not provide control of the output light distribution and provide only Lambertian diffusion. Referring then to Figures 9 - 12, there is shown a pair of light distribution patterns and a corresponding three-dimensional surface structure for producing the light distribution pattern. More particularly, with reference to Figure 9 a circular light distribution is shown having an angular spread φ on the order of 1-10 degrees and more preferably, about 2-3 degrees. The corresponding structure is shown in the photograph of Figure 10. In contrast, the wider-angle (φ equal about 2 degrees by φ equal about 80 degrees) elliptical shaped light distribution pattern is shown in Figure 11. The corresponding surface structure is shown in Figure 12. Selection of an appropriate three-dimensional surface structure will depend on a number of factors including the sample to be prepared, the analysis technique and analysis equipment, including detector size and type, being used.

Two important characteristics of the three-dimensional surface structure are the aspect ratio, i.e. , the ratio of the depth to the pitch of the surface structures and the apex ratio, i.e., the ratio of the depth to the diameter of the surface structures. The dimensional characteristics of the surface structures are respectively illustrated for a circular structure in Figure 14A and for an elliptical structure in Figure 14B.. For example, an aspect ratio in the range of 1: 1 to 1 :6 (depth to pitch), and an apex ratio (depth to diameter) in the range of 1:0.5 to 1: 1 may be used. Certain aspect and apex ratios should be selected based upon the application. For illustration only, examples of preferred aspect and apex ratios for a number of structures are set forth in Table I. It will be appreciated that the three-dimensional structures are totally random, and therefore, precise measurements are not possible. The preferred aspect and apex ratios of a structure are thus estimated by measuring a number of the structures. It will also be appreciated that other larger or smaller aspect and apex ratios may be used, but manufacturing limitations currently limit larger aspect ratios. A preferred arrangement may utilize surface structures on the order of about 1-5 microns high by 2-10 microns wide for cells but preferably smaller for molecules. Larger or smaller structures may be used. In either case, optimal surface structure is dependent on the sample, and the aspect ratio is selected to maximize surface area for the sample of interest. Such control and optimization of the surface structure and surface area was heretofore not possible. Moreover, the three- dimensional surface structure may be formed with a substantially random orientation that is beneficial to sample growth.

Table I: Dimensional Characteristics of Light Shaping Structures

2. Uses of Apparatus Incorporating Three-dimensional Structures A. Concentrators

Due to the adherent properties of the diffuser structure, it can also be used as a concentrator of chemicals. For example, many assays detect a small amount of a chemical. Accordingly, a method to first concentrate a sample before the detection is desirable. Concentrations may be performed with the diffuser structure itself.

Additionally, samples can be concentrated either (1) in a regular, closed structure, such as those illustrated in Figs. 1, 3, and 6, or (2) in a structure with at least one passageway, as illustrated in Fig. 15. In a regular, closed structure, the sample is added to the well or other appropriate apparatus, such as a flask, and the chemical of interest in the sample is allowed to adhere to the diffuser structure. The sample fluid is then removed and replaced with a new sample. Again, the chemical of interest is allowed to adhere, and then the sample fluid is removed. This application of sample, adherence, and aspiration are repeated for the desired number of times. Next, the chemical of interest may be detected, if so desired. Additionally, the chemical of interest may be quantified. Alternatively, if no detection is desired, but only concentration is desired, the method ends.

To use the diffuser as a concentrator in an apparatus having a passageway, the sample is passed through the structure either continuously or intermittently. The passageway may then be closed to add new fluid, with the new fluid being a plurality of fluids, including more samples. Alternatively, the new fluid may be dyes or other chemicals that could be used to detect the adhered chemicals. Yet still another alternative is that no detection would take place. Instead, the new fluid may contain a reagent from a protocol that is added to the concentrated chemicals. Additionally, concentration in and of itself may be the desired endpoint.

The diffuser structure may also be precoated with chemicals. Again, either the regular structure or the structure containing the passageway may be used.

Many changes and modifications could be made to the invention without departing from the fair scope and spirit thereof. The scope of some changes is discussed above. The scope of others will become apparent from the appended claims.

Claims

CLAIMS We claim:

1. An apparatus for analyzing a sample comprising: a material having a surface on which said sample is placed, said surface formed to include a three-dimensional light shaping structure.

2. An apparatus of claim 1, wherein said apparatus is a monolith.

3. An apparatus of claim 1, wherein said apparatus is a microtiter plate.

4. An apparatus of claim 1, wherein said apparatus is an analysis chamber.

5. A plate for analyzing a sample comprising: a monolith of substantially optically transparent material, said monolith having a top surface on which said sample is placed and a bottom surface, said top surface formed to include a three-dimensional light shaping structure.

6. A plate of claim 5, said light-shaping structure arranged to enhance sample adhesion.

7. A plate of claim 5, said light-shaping structure comprising a plurality of microstructures formed in said top surface.

8. A plate of claim 7, wherein each said microstructure has an aspect ratio of about 1:3.

9. A plate of claim 7, wherein each said microstructure has an aspect ratio of about 1:2.

10. A plate of claim 7, wherein each said microstructure has a height in the range of about 1-5 microns.

11. A plate of claim 7, said plurality of microstructures arranged to provide a circular light distribution having an angular spread of approximately 80 degrees.

12. A plate of claim 7, said plurality of microstructures arranged to provide an approximately 2 degrees by 80 degrees angular light distribution.

13. A plate of claim 7, said plurality of microstructures arranged to provide a substantially circular light distribution.

14. A plate of claim 5, said light shaping structure having a gradient index of refraction extending into said monolith.

15. A plate of claim 5, said light shaping structure reducing backscatter.

16. A plate of claim 5, said bottom surface formed to include a second light shaping structure.

17. A microtiter plate comprising: a substantially optically transparent substrate, said substrate having a first surface and a second surface; and a plurality of analysis wells formed into said first surface, each said analysis well having a sidewall and a bottom defining an analysis chamber, and wherein said bottom is formed to include a three-dimensional light shaping structure.

18. The microtiter plate of claim 17, wherein each analysis well has a shape selected from the group of shapes consisting of circular, rectangular, rounded rectangular, triangular, hexagonal, parabolic, inverse parabolic, U- bottom, and N-bottom.

19. The microtiter plate of claim 17, each said light shaping structure arranged to enhance sample adhesion.

20. The microtiter plate of claim 17, each said light shaping structure comprising a plurality of microstructures formed in said bottom.

21. The microtiter plate of claim 20, wherein each said microstructure has an aspect ratio in the range of about 1:3.

22. The microtiter plate of claim 20, wherein each said microstructure has an aspect ratio of about 1:2.

23. The microtiter plate of claim 20, wherein each said microstructure has a height in the range of about 1-5 microns.

24. The microtiter plate of claim 20, each said plurality of microstructures arranged to provide an angular light distribution in the range of approximately 2 degrees by 10 degrees to about 2 degrees by 80 degrees.

25. The microtiter plate of claim 20, each said plurality of microstructures arranged to provide approximately 2 degrees by 80 degrees angular light distribution.

26. The microtiter plate of claim 20, each said plurality of microstructures arranged to provide a substantially circular light distribution.

27. The microtiter plate of claim 19, each said light shaping structure having a gradient index of refraction extending into said substrate.

28. The microtiter plate of claim 19, each said light shaping structure reducing backscatter.

29. The microtiter plate of claim 19, each said second surface formed to include a second light shaping structure.

30. A microtiter plate comprising: a monolith, said monolith having a top surface and a bottom surface; and a well array, said well array having a first side and a second side and a plurality of passages formed therein, each passage having a first open end and a second open end and a sidewall, and each passage formed to include a three- dimensional light shaping structure; said well array being securable to said top surface such that when said well array is secured to said top surface each passage is closed and when said well array is not secured to said top surface each passage is open.

31. The microtiter plate of claim 30, wherein each passage has a shape selected from the group of shapes consisting of circular, rectangular, rounded rectangular, triangular, hexagonal, parabolic, and inverse parabolic.

32. The microtiter plate of claim 30, wherein each said light shaping structure is arranged to enhance molecular adhesion.

33. The microtiter plate of claim 30, wherein each said light shaping structure comprises a plurality of microstructures formed in said sidewall.

34. The microtiter plate of claim 33, wherein each said microstructure has an aspect ratio of about 1:3.

35. The microtiter plate of claim 33, wherein each said microstructure has an aspect ratio of about 1:2.

36. The microtiter plate of claim 33, wherein each said microstructure has a height in the range of about 1-5 microns.

37. The microtiter plate of claim 33, each said plurality of microstructures arranged to provide an angular light distribution in the range of approximately 2 degrees by 10 degrees to about 2 degrees by 80 degrees.

38. The microtiter plate of claim 33, each said plurality of microstructures arranged to provide approximately 2 degrees by 80 degrees angular light distribution.

39. The microtiter plate of claim 33, each said plurality of microstructures arranged to provide a substantially circular light distribution.

40. The microtiter plate of claim 30, each said light shaping structure having a gradient index of refraction extending into said monolith.

41. The microtiter plate of claim 30, each said light shaping structure reducing backscatter.

42. The microtiter plate of claim 30, each said bottom surface formed to include a second light shaping structure.

43. An analysis flask comprising: a monolith of substantially optically transparent material, said monolith having a top surface and a bottom surface, said top surface formed to include a three-dimensional light shaping structure; and an analysis chamber secured to said monolith and over said top surface and defining a fluid tight chamber.

44. The analysis flask of claim 43, further comprising a sample inlet port formed in said analysis chamber.

45. The analysis flask of claim 43, said light-shaping structure arranged to enhance molecular adhesion.

46. The analysis flask of claim 43, said light-shaping structure comprising a plurality of microstructures formed in said top surface.

47. The analysis flask of claim 46, wherein each said microstructure has an aspect ratio in the range of about 1:3.

48. The analysis flask of claim 46, wherein each said microstructure has an aspect ratio of about 1:2.

49. The analysis flask of claim 46, wherein each said microstructure has a height in the range of about 1-5 microns.

50. The analysis flask of claim 46, said plurality of microstructures arranged to provide an angular light distribution in the range of approximately 2 degrees by 10 degrees to about 2 degrees by 80 degrees.

51. The analysis flask of claim 46, said plurality of microstructures arranged to provide an approximately 2 degrees by 80 degrees angular light distribution.

52. The analysis flask of claim 46, said plurality of microstructures arranged to provide a substantially circular light distribution.

53. The analysis flask of claim 43, said light shaping structure having a gradient index of refraction extending into said monolith.

54. The analysis flask of claim 43, said light shaping structure reducing backscatter.

55. The analysis flask of claim 43, said bottom surface formed to include a second light shaping structure.

56. An analysis chamber comprising: a substantially optically transparent substrate, said transparent substrate having a first surface and a second surface; and said transparent substrate having a passageway from said first surface to said second surface, wherein said passageway is formed to include a three-dimensional light shaping structure.

57. A method comprising the steps of: providing a substantially optically transparent substrate, said substrate having a first surface and a second surface; placing a sample into at least one of a plurality of analysis wells formed in said first surface, each said analysis well having a sidewall and a bottom defining an analysis chamber, and wherein said bottom is formed to include a three-dimensional light shaping structure; and adhering said sample to said three-dimensional light shaping structure.

58. A method as defined in claim 57, further comprising: illuminating said substrate and said sample in said analysis wells with light from a light source, said light shaping structure shaping light emanating from said sample into a desired light output distribution; and detecting said desired light output distribution.

59. A method as defined in claim 57, wherein said sample comprises a chemical.

60. A method as defined in claim 59, wherein a desired light distribution is circular and has an angular spread in the range of approximately 20 degrees to 80 degrees.

61. A method as defined in claim 59, wherein a light distribution is circular and has an angular spread of approximately 80 degrees.

62. A method for concentrating a sample comprising the steps of: providing a substantially optically transparent substrate, said transparent substrate having a first surface and a second surface; and said transparent substrate having a passageway from said first surface to said second surface, wherein said passageway is formed to include a three-dimensional light shaping structure; placing said sample into at least one of a plurality of analysis wells formed in said first surface, each said analysis well having a sidewall and a bottom defining an analysis chamber, and wherein said bottom is formed to include said three- dimensional light shaping structure; adhering said sample to said three-dimensional light shaping structure; and allowing said sample not adhered to said structure to flow through said passageway.

63. A method as defined in claim 62, wherein said passageway is pre-adhered with a first chemical.

64. A method for concentrating a sample comprising the steps of: providing a substantially optically transparent substrate, said substrate having a first surface and a second surface; placing said sample into at least one of a plurality of analysis wells formed into said first surface, each said analysis well having a sidewall and a bottom defining an analysis chamber, and wherein said bottom is formed to include a three- dimensional light shaping structure; and adhering said sample to said three-dimensional light shaping structure.

65. A method as described in claim 64, further comprising removing said sample not adhered to said structure.

66. A method as described in claim 65, further comprising repeating said placing, adhering, and removing steps.

67. A method as defined in claim 64, wherein said structure is pre-adhered with a first chemical.

68. A method of analyzing a sample, said method comprising the steps of: providing a substantially optically transparent substrate, said substrate having a first surface and a second surface; placing said sample into at least one of a plurality of analysis wells formed in said first surface, each said analysis well having a sidewall and a bottom defining an analysis chamber, and wherein said bottom is formed to include a three-dimensional light shaping structure; and detecting a chemical of interest.

69. A method as defined in claim 68, further comprising illuminating said substrate and said sample in said analysis wells with light from a light source, said light shaping structure reducing backscatter of light incident said substrate and said sample and shaping light emanating from said sample into a desired light distribution; and detecting said desired light output distribution.

70. A method as defined in claim 68 wherein said desired light distribution is circular and has an angular spread in the range of 1 degrees to 10 degrees.

71. A method as defined in claim 70 wherein said light distribution has an angular spread of approximately 2 degrees to 3 degrees.

72. A method as defined in claim 68, wherein said three-dimensional light shaping structure is pre-adhered with a first chemical.

73. An apparatus for analyzing a sample comprising a material having a first substantially planar side and a second substantially planar side and at least one of said first substantially planar side and said second substantially planar side formed to include a plurality of randomly distributed facets, each facet of said plurality of randomly distributed facets has a size approximate a size of said sample.

74. The apparatus of claim 73, said material being translucent.

75. The apparatus of claim 73, wherein each facet of said plurality of randomly distributed facets has an aspect ratio of about 1:2.

76. The apparatus of claim 73, wherein each facet of said plurality of randomly distributed facets has an aspect ratio of about 1:3.

77. The apparams of claim 73, wherein said aspect ratio is optimized for surface area.

78. The apparatus of claim 73, wherein said apparams is a plate.

79. The apparams of claim 73, wherein said apparams is a microtiter plate.

80. The apparams of claim 79, said microtiter plate comprising a parallel assemblage of upstanding tubes and a base plate, said tubes bonded to said base plate, said base plate having a surface structure to homogenize light with directionality and to control light propagation.

81. The apparams of claim 80, said surface structure comprising said random distribution of facets.

82. The apparatus of claim 79, said microtiter plate comprising a parallel assemblage of upstanding tubes and a base plate, said tubes bonded to said base plate, so as to form an essentially fluid tight chamber, and said at least one of said first substantially planar side and said second substantially planar side forming a bottom of said chamber.

83. The apparams of claim 73, said apparatus comprising an analysis flask.

84. The apparams of claim 83, said analysis flask having a closed bottom portion and an open top portion, and said at least one of said first substantially planar side and said second substantially planar side forming said bottom portion.