GB2496315A - Multiplex optical assembly - Google Patents

Abstract

An imaging system for imaging a plurality of reagent beads retained in a sample well of a sample plate is disclosed comprising a collection lens assembly positioned between a non-Fresnel field lens and a camera sensor array. The field lens assembly may comprise a negative meniscus lens 1. The collection lens assembly may comprise in order a plano-convex lens 2, a biconvex lens 3, a biconcave lens 4, a plano­concave or biconcave lens 5, a biconvex lens 6 and a negative meniscus lens 7. A further embodiment relates to a luminometer of the same configuration for analyzing luminescent samples. Use of this arrangement allows for a relatively short optical path length between the field lens and the collection lens assembly, allowing for smaller instrument size while still producing an even optical response across the imaging field of the sample plate.

Description

MULTIPLEX OPTICAL ASSEMBLY

BACKGROUND TO THE PRESENT INVENTION

The present invention relates to an multiplex optical assembly and in particular an imaging device for analysing a plurality of reagent beads retained in a sample plate. The preferred embodiment relates to a lens system for a multiplexing platform which reduces the overall optical path length, minimizes crosstalk, establishes a relatively flat response across the sample plate and optimizes sensitivity.

Immunoassay procedures are a preferred way of testing biological products. These procedures exploit the ability of antibodies produced by the body to recognise and combine with specific antigens which rriay, for example, be associated with foreign bodies such as bacteria or viruses, or with other body products such as hormones. Once a specific antigen-antibody combination has occurred it can be detected using chromogenic.

fluorescent or chemiluminescent materials or less preferably by using radioactive substances. Radioactive substances are less preferred due to environmental and safety concerns regarding their handling, storage and disposal. The same principles can be used to detect or determine any materials which can form specific binding pairs, for example using lectins, rheumatoid factor, protein A or nucleic acids as one of the binding partners.

Enzyme-linked Immunosorbent Assay ("ELISA") is a particularly preferred form of immunoassay procedure wherein one member of the binding pair is linked to an insoluble carrier surface ("the solid phase") such as a sample vessel, and after reaction the bound pair is detected by use of a further specific binding agent conjugated to an enzyme ("the conjugate"). The procedures for ELISA are well known in the art and have been in use for both research and commercial purposes for many years. Numerous books and review articles describe the theory and practice of immunoassays. Advice is given, for example, on the characteristics and choice of solid phases for capture assays, on methods and reagents for coating solid phases with capture components, on the nature and choice of labels, and on methods for labelling components. An example of a standard textbook is "ELISA and Other Solid Phase Immunoassays, Theoretical and Practical Aspects", Editors D.M. Kemeny & S.J. Challacombe, published by John Wiley, 1988. Such advice may also be applied to assays for other specific binding pairs.

In the most common type of ELISA, the solid phase is coated with a member of the binding pair. An aliquot of the specimen to be examined is incubated with the solid coated solid phase and any analyte that may be present is captured onto the solid phase. After washing to remove residual specimen and any interfering materials it may contain, a second binding agent, specific for the analyte and conjugated to an enzyme is added to the solid phase. During a second incubation any analyte captured onto the solid phase will combine with the conjugate. After a second washing to remove any unbound conjugate, a chromogenic substrate for the enzyme is added to the solid phase. Any enzyme present will begin to convert the substrate to a chromophoric product. After a specified time the amount of product formed may be measured using a spectrophotometer, eithei directly or after stopping the reaction.

It will be realised that the above is an outline description of a general procedure for bioassay and that many variants are known in the art including fluorogenic and luminogenic substrates for ELISA, direct labelling of the second member of the binding pair with a fluorescent or luminescent molecule (in which case the procedure is not called an ELISA but the process steps are very similar) and nucleic acids or other specific pairing agents instead of antibodies as the binding agent. However, all assays require that fluid samples, e.g. blood, serum, urine, etc., are aspirated from a sample tube and are then dispensed into a solid phase. Samples may be diluted prior to being dispensed into the solid phase or they may be dispensed into deep well microplates, diluted in situ and then the diluted analyte may be transferred to the functional solid phase.

The most common type of solid phase is a standard sample vessel known as a microplate which can be stored easily and which may be used with a variety of biological specimens. Microplates have been available commercially since the 1 960s and are made from e.g. polystyrene, PVC, Perspex or Lucite and measure approximately 5 inches (12.7 cm) in length, 3.3 inches (8.5 cm) in width, and 0.55 inches (1.4 cm) in depth. Microplates made from polystyrene are particularly preferred on account of polystyrene's enhanced optical clarity which assists visual interpretation of the results of any reaction. Polystyrene microplates are also compact, lightweight and easily washable. Microplates manufactured by the Applicants are sold under the name "MICROTITRE" (RIM). Known microplates comprise 96 wells (also commonly known as "microwells") which are symmetrically arranged in an 8 x 12 array. Microwells typically have a maximum volume capacity of approximately 350 p1. However, normally only 10-200 p1 of fluid is dispensed into a microwell. In some arrangements of the microplate the microwells may be arranged in strips of 8 or 12 wells that can be moved and combined in a carrier to give a complete plate having conventional dimensions.

Positive and negative controls are generally supplied with commercial kits and are used for quality control and to provide a relative cut-off. After reading the processed microplate, the results of the controls are checked against the manufacturer's validated values to ensure that the analysis has operated correctly and then the value is used to distinguish positive from negative specimens and a cut-off value is calculated. Standards are usually provided for quantitative assays and are used to build a standard curve from which the concentration of analyte in a specimen may be interpolated.

It will be recognised that the ELISA procedure as outlined above involves multiple steps including pipetting, incubation, washing, transferring microplates between activities, reading and data analysis. In recent years systems have been developed which automate the steps (or "phases") involved in the ELISA procedures such as sample distribution, dilution, incubation at specific temperatures, washing, enzyme conjugate addition, reagent addition, reaction stopping and the analysis of results. The pipette mechanism used to aspirate and dispense fluid samples uses disposable tips which are ejected after being used so as to prevent cross-contamination of patients' samples. Multiple instrumental controls are in place to ensure that appropriate volumes, times, wavelengths and temperatures are employed, data transfer and analysis is fully validated and monitored.

Automated immunoassay apparatus for carrying out ELISA procedures are now widely used in laboratories of e.g. pharmaceutical companies, veterinary and botanical laboratories, hospitals and universities for in-vitro diagnostic applications such as testing for diseases and infection, and for assisting in the production of new vaccines and drugs.

ELISA kits are commercially available which consist of microplates having microwells which have been coated by the manufacturer with a specific antibody (or antigen). For example, in the case of a hepatitis B antigen diagnostic kit, the kit manufacturer will dispense anti-hepatitis B antibodies which have been suspended in a fluid into the microwells of a microplate. The microplate is then incubated for a period of time, during which time the antibodies adhere to the walls of the microwells up to the fluid fill level (typically about half the maximum fluid capacity of the microwell). The microwells are then washed leaving a microplate having microwells whose walls are uniformly covered with anti-hepatitis B antibodies up to the fluid fill level.

A testing laboratory will receive a number of sample tubes containing, for example, body fluid from a number of patients. A specified amount of fluid is then aspirated out of the sample tube using a pipette mechanism and is then dispensed into one or more microwells of a microplate which has been previously prepared by the manufacturer as discussed above. If it is desired to test a patient for a number of different diseases then fluid from the patient must be dispensed into a number of separate microplates, each coated by its manufacturer with a different binding agent. Each microplate can then be processed separately to detect the presence of a different disease. It will be seen that to analyse several different analytes requires a multiplicity of microplates and transfer of aliquots of the same specimen to the different microplates. This leads to large numbers of processing steps and incubators and washing stations that can cope with many microplates virtually simultaneously. In automated systems this requires instruments to have multiple incubators and complex programming is required to avoid clashes between microplates with different requirements. For manual operation either several technicians are required or the throughput of specimens is slow. It is possible to combine strips of differently coated microwells into a single carrier, add aliquots of a single specimen to the different types of well and then perform the ELISA in this combined microplate. Constraints on assay development! however, make this combination difficult to achieve and it is known in the art that for users to combine strips in this fashion can lead to errors of assignment of result, while manufacture of niicroplates with several different coatings in different microwells presents difficulties of quality control.

Conventional ELISA techniques have concentrated upon performing the same single test upon a plurality of patient samples per microplate or in detecting the presence of one or more of a multiplicity of analytes in those patients without distinguishing which of the possible analytes is actually present. For example, it is commonplace to determine in a single microwell whether a patient has antibodies to HIV-1 or HIV-2, or HIV-1 or -2 antigens, without determining which analyte is present and similarly for HCV antibodies and antigens.

However, a new generation of assays are being developed which enable multiplexing to be performed. Multiplexing enables multiple different tests to be performed simultaneously upon the same patient sample.

A recent approach to multiplexing is to provide a microplate comprising 96 sample wells wherein an array of different capture antibodies is disposed in each sample well. The array comprises an array of 20 nI spots each having a diameter of 350 pm. The spots are arranged with a pitch spacing of 650 pm. Each spot corresponds with a different capture antibody.

Multiplexing enables a greater number of data points and more information per assay to be obtained compared with conventional ELISA techniques wherein each sample plate tests for a single analyte of interest. The ability to be able to combine multiple separate tests into the same assay can lead to considerable time and cost savings.

Multiplexing also enables the overall footprint of the automated apparatus to be reduced.

In addition to ELISA procedures it is also known to use a hybridization probe to test for the presence of DNA or RNA sequences. A hybridization probe typically comprises a fragment of DNA or RNA which is used to detect the presence of nucleotide sequences which are complementary to the DNA or RNA sequence on the probe. The hybridization probe hybridizes to single-stranded nucleic acid (e.g. DNA or RNA) whose base sequence allows pairing due to complementarity between the hybridization probe and the sample being analysed. The hybridization probe may be tagged or labelled with a molecular marker such as a radioactive or more preferably a fluorescent molecule. The probes are inactive until hybridization at which point there is a conformational change and the molecule complex becomes active and will then fluoresce (which can be detected under UV light) DNA sequences or RNA transcripts which have a moderate to high sequence similarity to the probe are then detected by visualising the probe under UV light.

GB-2472882 discloses a sample plate comprising a plurality of sample wells. Each sample well comprises a plurality of pockets into which reagent beads or macrospheres are inserted by a reagent bead or macrosphere dispenser. The reagent beads or macrospheres may be coated with an antibody or antigen enabling multiplexed ELISA procedures to be performed. Alternatively, the reagent beads may be coated with a DNA or RNA sequence to act as a hybridization probe to test for the presence of a complementary DNA or RNA sequence.

The sample plate disclosed in GB-2472882 has been further developed and sample plates are known having cylindrical through holes wherein reagent beads are inserted into the through holes and are retained by a circumferential interference fit with the through hole.

Luminometers for monitoring light emitting reactions are known. Known luminometers use one or more photomultiplier tubes (PMTs) to detect the photons emitted.

Other optical detection systems are known but which use complex teleconcentric glass lenses. Such systems suffer from the problem that they provide a distorted view of sample wells at the edges of the sample plate.

US2O1O/0248387 discloses a luminescence detecting apparatus and a method of analysing luminescent samples. Luminescent samples are placed in a chamber. Light from the luminescent samples passes through a collimator, a Fresnel field lens, a filter and a camera lens. A focused image is created by the optics on a charge-coupled device (CCD") camera. The disclosed optical system includes a dark collimator which permits only parallel or semi-parallel light rays to exit the sample wells for eventual imaging by the CCD camera.

As will be discussed in more detail below, the use of a Fresnel field lens is particularly problematic.

Designing an optical detection system for optically detecting and reading reagent beads located in the wells of a sample plate is particularly problematic. An approach to an optical detection system requires an optical path length of approximately 1 m for the light emitted from some spot (i.e. reagent bead) positions to be collected properly. Experiments have been performed using a single commercial imaging lens. However, the use of a single commercially available lens for the imaging lens suffers from a number of problems.

Firstly, the optical path length needs to be approximately 1 m long which is impractical for a commercial clinical analyzer mechanical design.

Secondly, the characteristics of the commercially available lens resulted in an uneven field being produced where the response fell off as much as 25% from the middle to the edges of the plate image.

Thirdly, another practical limitation to using a long overall clear path with a single custom designed lens with a relatively flat field is that the lens has to be relatively large (e.g. 125 mm CD or greater) and the cost of such commercial lenses is in excess of $4,000. Such a solution is therefore impractical for a commercial imaging system.

It is therefore desired to provide an improved imaging system for analysing a plurality of reagent beads retained in a sample plate.

SUMMARY OF THE PRESENT INVENTION

According to an aspect of the present invention there is provided an imaging system for imaging a plurality of reagent beads retained in a sample well of a sample plate, comprising:

a non-Fresnel field lens or field lens assembly;

a collection lens assembly; and a camera sensor array; wherein the collection lens assembly is positioned between the non-Fresnel field lens or field lens assembly and the camera sensor array.

The non-Fresnel field lens or field lens assembly preferably comprises at least a

first field lens and a second field lens.

The first field lens is preferably selected from the group consisting of: (i) a bi-convex lens; (ii) a piano-convex lens; (iii) a convex-meniscus lens; (iv) a bi-concave lens; (v) a piano-concave lens; and (vi) a concave-meniscus lens.

The second field lens is preferably selected from the group consisting of: (i) a bi-convex lens; (ii) a piano-convex lens; (iii) a convex-meniscus lens; (iv) a bi-concave lens; (v) a piano-concave lens; and (vi) a concave-meniscus lens.

The first field lens preferably comprises a bi-convex, piano-convex or convex- meniscus lens and the second field lens comprises bi-convex, piano-convex or convex-meniscus lens.

The collection lens assembly preferably comprises at least a first collection lens and a second collection lens.

The first collection lens is preferably selected from the group consisting of: (i) a bi-convex lens; (U) a piano-convex lens; (Di) a convex-meniscus lens; (iv) a bi-concave lens; (v) a piano-concave lens; and (vi) a concave-meniscus lens.

The second collection lens is preferably selected from the group consisting of: (i) a bi-convex lens; (ii) a piano-convex lens; (iii) a convex-meniscus lens; (iv) a bi-concave lens; (v) a piano-concave lens; and (vi) a concave-meniscus lens.

Light from the reagent beads preferably passes, in use, from the first collection lens to the second collection lens, wherein the first collection lens comprises a bi-convex, piano-convex or convex-meniscus lens and the second collection lens comprises a bi-concave, piano-concave or concave-meniscus lens.

The imaging system preferably further comprises a third collection lens wherein light from the reagent beads passes, in use, from the first collection lens to the second collection lens, then from the second collection lens to the third collection lens, wherein the third collection lens comprises a bi-convex, piano-convex or convex-meniscus lens.

The imaging system preferably further comprises a fourth collection lens wherein light from the reagent beads passes, in use, from the first collection lens to the second collection lens, then from the second collection lens to the third collection lens, then from the third collection lens to the fourth collection lens, wherein the fourth collection lens comprises a bi-convex, piano-convex or convex-meniscus lens.

The collection lens assembly preferably has a F/# selected from the group consisting of: (i) F/O.70 -Ff0.80; (ii) Ff0.80-F/0.90; (Hi) Ff0.90-F/i.00; and (iv) F/i.00 -Ff1.10.

The non-Fresnel field lens or field lens assembly and/or the collection lens assembly preferably has a magnification selected from the group consisting of: (i) C 0.12; (ii) 012-0.14; (iH) 0.14-0.16; (iv) 0.16-0.18; (v)0.i8-0.20; and (vi) > 0.20.

An optical path length from a base portion of the sample well of the sample plate to the detecting surface of the camera sensor array is preferably selected from the group consisting of: (i) 50-100 mm; (ii) 100-150 mm; (iii) 150-200 mm; (iv) 200-250 mm; (v)250- 300 mm; (vi) 300-350 mm; (vii) 350-400 mm; and (viH) 400-450 nm; (ix) 450-500 nm.

The camera sensor array preferably comprises a charge coupled device (CCD) camera.

According to an aspect of the present invention there is provided macroarrayer comprising an imaging system as described above.

The macroarrayer preferably further comprising a sample plate.

The sample well of the sample plate preferably has an internal diameter selected from the group consisting of: (I) 5-6 mm; (ii) 6-7 mm; (iii) 7-8 mm; (iv) 8-9 mm; (v) 9-10 mm; (vi) 10-11 mm; (vU) 11-12 mm; (vUi) 12-13 mm; (ix) 13-14 mm; (x) 14-15 mm; (xi) 15-16 mm; (xU) 16-17 mm; (xih) 17-18 mm; (xiv) 18-19 mm; and (xv) 19-20 mm.

The reagent beads or macrobeads preferably have a diameter selected from the group consisting of: (0< 0.5 rpm; (ii) 0.5-1.0 mm; (iii) 1.0-1.5 mm; (iv) 1.5-2.0 rpm; (v)2.0- 2.5 mm; (vi) 2.5-3.0 mm; (vU) 3.0-3.5 mm; (viii) 3.5-4.0 mm; (ix) 4.0-4.5 mm; and (x) 4.5-5.0 mm.

The sample well preferably has a base portion having a plurality of blind recesses or through-holes, wherein a reagent bead is inserted or retained in each of the blind recesses or through-holes and wherein the depth of the sample well as measured from an opening into the sample well through which sample is dispensed in use to the base portion is selected from the group consisting of: (i) 2-3 mm; (ii) 3-4 mm; (iii) 4-5 mm; (iv) 5-6 mm; (v) 6-7 mm; (vi) 7-8 mm; (vU) 8-9 mm; and (vUi) 9-10 mm.

The macroarrayer preferably further comprises a processing system, the processing system being arranged and adapted to detect and quantify luminescence of reagent beads in the sample plate as an indicator of the presence or amount of a target compound.

Light is preferably emitted, in use, from the reagent beads due to bioluminescence or chemiluminescence and has a peak in a range selected from the group consisting of: (i) 400-410 nm; (ii) 410-420 nm; (iii) 420-430 nm; (iv) 430-440 nm; (v)440-450 nm; (vi) 450- 460 nm; (vii) 460-470 nm; (vUi) 470-480 nm; (ix) 480-490 nm; and (x) 490-500 nm.

According to an aspect of the present invention there is provided a luminometer for analysing a plurality of luminescent samples comprising:

a non-Fresnel field lens or field lens assembly;

a collection lens assembly; and a camera sensor array; the collection lens assembly being positioned between the non-Fresnel field lens or

field lens assembly and the camera sensor array.

The plurality of luminescent samples preferably comprise bioluminescent or cherniluminescent samples.

According to an aspect of the present invention there is provided a method of imaging a plurality of reagent beads retained in a sample well of a sample plate, comprising: providing a non-Fresnel field lens or field lens assembly, a collection lens assembly and a camera sensor array, wherein the collection lens assembly is positioned between the non-Fresnel field lens or field lens assembly and the camera sensor array; and using the camera sensor array to determiner the intensity of the reagent beads.

According to an aspect of the present invention there is provided an imaging system for imaging a plurality of reagent beads retained in a sample well of a sample plate, comprising: a non-Fresnel field lens or field lens assembly, the non-Fresnel field lens or field lens assembly comprising a first bi-convex, piano-convex or convex-meniscus lens and a second bi-convex, piano-convex or convex-meniscus lens; a collection lens assembly, the collection lens assembly comprising a first, second, third and fourth collection lens, wherein light from the reagent beads passes, in use, from the first collection lens to the second collection lens, then from the second collection lens to the third collection lens, then from the third collection lens to the fourth collection lens, wherein the first, third and fourth collection lenses comprise bi-convex, piano-convex or convex-meniscus lenses and the second collection lens comprises a bi-concave, piano-concave or concave-meniscus lens; and a camera sensor array; wherein the collection lens assembly is positioned between the non-Fresnel field lens or field lens assembly and the camera sensor array.

According to an aspect of the present invention there is provided a method of imaging a plurality of reagent beads retained in a sample well of a sample plate, comprising: providing a non-Fresnel field lens or field lens assembly, the non-Fresnel field lens or field lens assembly comprising a first bi-convex, piano-convex or convex-meniscus lens and a second bi-convex, piano-convex or convex-meniscus lens; providing a collection lens assembly, the collection lens assembly comprising a first, second, third and fourth collection lens, wherein light from the reagent beads passes, in use, from the first collection lens to the second collection lens! then from the second collection lens to the third collection lens, then from the third collection lens to the fourth collection lens, wherein the first, third and fourth collection lenses comprise bi-convex, pIano-convex or convex-meniscus lenses and the second collection lens comprises a bi-concave, piano-concave or concave-meniscus lens; providing a camera sensor array, wherein the collection lens assembly is positioned between the non-Fresnel field lens or field lens assembly and the camera sensor array; and using the camera sensor array to determine the intensity of the reagent beads.

According to an aspect of the present invention there is provided an imaging system is for imaging a plurality of reagent beads retained in a sample well of a sample plate comprising:

a non-Fresnel field lens or field lens assembly;

a collection lens assembly; and a camera sensor array; wherein the collection lens assembly is positioned between the non-Fresnel field lens or field lens assembly and the camera sensor array.

The field lens or field lens assembly preferably comprises a negative meniscus lens.

The collection lens assembly preferably comprises a first lens.

The first lens preferably comprises a piano-convex lens.

The collection lens assembly preferably comprises a second lens wherein light from the reagent beads passes, in use, from the first lens to the second lens.

The second lens preferably comprises a biconvex lens.

The collection lens assembly preferably comprises a third lens wherein light from the reagent beads passes, in use, from the second lens to the third lens.

The third lens preferably comprises a biconcave lens.

The collection lens assembly preferably comprises a fourth lens wherein light from the reagent beads passes, in use, from the third lens to the fourth lens.

The fourth lens preferably comprises a piano-concave or biconcave lens.

The collection lens assembly preferably comprises a fifth lens wherein light from the reagent beads passes, in use, from the fourth lens to the fifth lens.

The fifth lens preferably comprises a biconvex lens.

The collection lens assembly preferably comprises a sixth lens wherein light from the reagent beads passes, in use, from the fifth lens to the sixth lens.

The sixth lens preferably comprises a negative meniscus lens.

According to an aspect of the present invention there is provided an imaging system for imaging a plurality of reagent beads retained in a sample well of a sample plate, comprising: a non-Fresnel field lens or field lens assembly comprising a negative meniscus lens; a collection lens assembly, the collection lens assembly comprising in order a first lens comprising a piano-convex lens, a second lens comprising a biconvex lens, a third lens comprising a biconcave lens, a fourth lens comprising a piano-concave or biconcave lens, a fifth lens comprising a biconvex lens and a sixth lens comprising a negative meniscus lens; and a camera sensor array; wherein the collection lens assembly is positioned between the non-Fresnel field lens or lens assembly and the camera sensor array.

According to an aspect of the present invention there is provided a method of imaging a plurality of reagent beads retained in a sample well of a sample plate, comprising: providing a non-Fresnel field lens or lens assembly comprising a negative meniscus lens; providing a collection lens assembly, the collection lens assembly comprising in order a first lens comprising a piano-convex lens, a second lens comprising a biconvex lens, a third lens comprising a biconcave lens, a fourth lens comprising a piano-concave or biconcave lens, a fifth lens comprising a biconvex lens and a sixth lens comprising a negative meniscus lens; and providing a camera sensor array, wherein the collection lens assembly is positioned between the non-Fresnel field lens or lens assembly and the camera sensor array; and using the camera sensor array to determine the intensity of the reagent beads.

According to an embodiment of the present invention one or two large diameter lenses may be used in close proximity to the test or sample plate. The large diameter type lenses are preferably field lenses which may comprise an assembly of lenses.

The purpose of the field lens(es) is to capture the emitted light from the beads retained in the sample plate and to direct the light towards collection imaging lenses.

An advantageous feature of the preferred embodiment is that light redirection by the field lens(es) reduces the optical path to the imaging collection lenses. According to an embodiment of the present invention the optical path may be reduced from approx. lm to 30-50 cm.

The preferred embodiment therefore represents a significant improvement compared with using a commercial imaging lens.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which:

Fig. 1 shows a prototype field lens layout;

Fig. 2 shows a test bead and well relationship; Fig. 3 shows a field lens and collection lens energy limitation; Fig. 4 shows some sample wells in which reagent beads are secured; Fig. 5 shows a test bench setup; Fig. 6 shows a preliminary 5 s exposure of a test plate;

Fig. 7 shows a Fresnel field lens;

Fig. 8 shows a view at a Fresnel surface and Fig. 8A shows rays transitioning between prisms; Fig. 9 shows test spot rays near the centre of the plate; Fig. 10 shows a view of slightly deviated light and undeviated light; Fig. iDA shows the passage of rays on a prism, Fig. lOB shows the focused rays in the image or sensor plane, Fig. 1OC shows five locations on a test spot (1 mm OD) and Fig. 100 shows all five spots shown in the sensor plane; Fig. 11 shows an example of a single radius field lens and Fig. 1 1A shows a MTF chart of Fig. 11 using a perfect 25 mm collection lens; Fig. 12 shows two off-the-shelf field lenses and Fig. 12A shows a MTF chart of the Fig. 12 configuration; Fig. 13 shows a single field lens with two powered surfaces and Fig. 13A shows a MTF chart of the Fig. 13 configuration; Fig. 14 shows a field lens configuration optimised with a collection lens and Fig. 14A shows the resolution of the combination; Fig. 15 shows an alternate field lens configuration with collection lens and Fig. iSA shows a MTF chart of the Fig. 15 configuration; and Fig. 16A shows lens elements according to a particularly preferred embodiment and Fig. lOB shows optical paths through the preferred lens elements.

A preferred embodiment of the present invention will now be described in more detail. Fig. 1 shows a prototype field lens comprising two lenses. The field lens is shown being located in close proximity to a sample plate which has a plurality of retained reagent beads. Five light emitting regions from the reagent beads are shown in Fig. 1.

Fig. 1 shows the emitted light from the sample plate on the lefthand side being collected and redirected by the two lenses forming the field lens. The field lens may comprise two piano-convex lenses. According to another embodiment the field lens may comprise a single negative meniscus lens.

Light emitted from the center of the test or sample plate does not need a field lens assembly to be directed toward a collection lens (not shown) which focuses the light onto a camera sensor array (not shown). In Fig. 1 the emitted light at the outermost position of the sample plate is shown being directed slightly outwards. There are projections of these outer positions that could be collected by the field lenses were it not for the further limiting factor that determines the direction and the light cone size, namely the depth and inside diameter of the multiplex wells.

Fig. 2 shows a schematic representation of three test beads located within a sample well of a sample plate. The center bead has a potentially unvignetted combination of smaller cones of light to be collected. The two end beads show limitations at the edges of the wells To be properly collected and directed to linearly proportional imaged locations, the central rays from these cones are height related. It Fig. 2 related to a well located in an extreme upper corner location on a test plate, then the central ray of the cones would be parallel to the mechanical axis. The direction of the central ray and the extreme ray would be just past the well edge determining one half the solid cone angle. This solid cone represents the potential energy flux that can be collected. The central location has a potentially larger cone of energy that is not blocked by the well height. It may be assumed that all of the potential cones of light are bent by the field lens assembly and are directed toward an imaging collection lens (not shown).

The imaging collection lens (not shown) is preferably arranged to have a fixed aperture that determines its F/number (F/#). The Ft# limits the collectable cone of energy from all locations on the test or sample plate. If the imaging collection lens were to be designed with an F/lU aperture, then some locations would have less than F/l.O cone of energy leaving the reagent bead and nearby locations would have more than Ff1.0. The result at the sensor in the image plane would be that the F/i.0 spots would be brighter than other spots that have some vignetting involved in determining the collectable cone of energy.

Fig. 3 shows aspects of an optical imaging system according to an embodiment of the present invention. Afield lens assembly comprising two lens is shown in close proximity to light emitting regions (i.e. sample beads) of a sample plate. The light from the sample plate is collected by the field lenses and is transmitted to a collection lens which images the light onto a camera detection system.

The right side of Fig. 3 shows a potential collection lens with an F/i.0 aperture.

Cones of light are shown behind the collection lens (on the righthand side of Fig. 3) and in front of the field lens assembly (on the lefthand side of Fig. 3). The ratio of these cones is the overall system magnification (the size of the sensor plane to the size of the multiplex plate). These are the theoretical parameters barring no vignetting.

Optical detection system without using a field lens assembly Referring to Fig. 3, if the two lenses forming the field lens were removed and the light cones from the multiplex sample plate were still projected as shown, then it is apparent that many locations would miss being collected. Referring back to Fig. 2, it can be seen there are potential cones of energy that have their central ray projected slightly downward. The projection would be in the direction of the F/if aperture. To achieve sufficient energy not blocked by the edge of the multiplex well the F/if aperture of the collection lens would have to be located at approximately 1 m distance.

An advantage of the preferred embodiment is that by using field lenses as shown in Fig. 3 the total axial length of the optical detection system can be reduced to e.g. 23.2 cm according to an embodiment (compared with 1 m without using field lenses).

Relationship between the field lens and the collection lens Referring to Fig. 3, attention is directed to the angles of the central rays leaving the field lens assembly. According to the preferred embodiment the rays are projected to intersect the F/if aperture of the downstream collection lens. The more optical bending (i.e. the shorter the focal length of the field lens), the closer the F/if aperture of the collection lens can be located to intersect these central rays.

As the separation between the field lens and the collection lens is reduced, both focal lengths have to change to maintain the desire optical magnification. The opposite is true when the separation increases.

Practical limitations for the separation between the field lens and the collection lens Commercially available collection lenses with low F/if's comprise FI0.95 lenses with mm and 50 mm EEL (focal lengths). The lenses are corrected for a 4/3" sensor format.

Commercially available field lenses that have a 6" OD have very limited focal lengths and must be used in combinations to achieve short separations.

From computer studies as shown by Fig. 3 where the collection lens is assumed to be perfect and the aberrations from the field lens combinations are examined in terms of potential resolution, test cases can be found.

Computer studies which were performed using a 50 mm EFL collection lens tended to have an overall length from the multiplex plate to the sensor plane of between 50-80 cm.

These computer studies were not developed further.

Computer studies involving using a 25 mm EEL lens were more promising as these enabled the overall length to be reduced to c 30 cm as in the particular example shown in Fig. 3.

The example shown in Fig. 3 was prototyped and the prototype showed advantageous results over a short overall length using a low F/ft collection lens.

It is possible with selection of combinations of field lenses to achieve even shorter overall lengths using 25 mm EFL lenses, but the optical magnification may suffer from the problem that the image overfills the camera sensor. In such cases the overall length could be reduced to 204 mm if a 21 mm EFL collection lens were commercially available. Any configuration shorter than this would require custom lenses which are expensive and therefore less preferred.

Fundamental optical design considerations using the multiplex test wells Fig. 2 is not an accurate representation of the placement of reagent beads in the sample wells of a sample plate. The purpose of Fig. 2 is merely to show how light exits and is limited by the well depth and bead placement.

Fig. 4 shows an accurate representation of a plurality of sample wells each having five through holes in the base portion into which reagent beads are inserted in use according to an embodiment. A central ray aiming toward a FI# aperture without the aid of field lenses can be approximated in order to show the effect of not using a field lens According to the particular embodiment shown in Fig. 4 the well depth to a bead location is 7.5 mm, the separation from the well wall to bead edge is 1 mm, and the smallest beads are 1.5 mm OD. These dimensions can be projected and result in the collection lens aperture stop being located at a distance of 350-450 mm. Testing with a commercial 25 mm, Ff0.95 lens near these dimensions still exhibits blocking and vignetting at the extreme locations of the test plate. When adding the lens barrel length and the camera length to these projected distances, the 450 mm length is extended to become 700 mm. To reach a working distance with no blocking/vignetting a larger distance would be needed. This is contrary to the need for a shorter optical path to be commercially viable.

Matching field lenses to a 25 mm commercially available collection imaging lens Fig. 3. shows an optical layout according to an embodiment of the present of the present invention which resulted from the analysis section of an optical design program.

The two large lenses which together comprise the field lens comprise standard commercial lenses. Their combination allowed the projected central rays to intersect a plane where a theoretical lens would exist. The optical parameters of the field lenses and a 25mm perfect collection imaging lens yields a separation of 150 mm between major components and the proper optical magnification where the test or sample plate is imaged on a 4/3" sensor.

Other combinations of off-the-shelf large OD lenses could not meet these conditions. The system shown in the optical layout was constructed to verify the prediction of a short -14-overall working distance and the 25 mm, F/0.95 collection lens could operate without blocking/vignetting.

Fig. 5 shows a prototype wherein a test or sample plate is located on the left. A cylinder can be seen which holds a field lens assembly in the center. An interface to a 25 mm EFL collection imaging lens and a camera detection system can be seen on the right.

A ruler shows that the overall distance from the test or sample plate to the back of the camera was approximately 300 mm. This corresponds with the computer model shown in Fig. 3. The physical length of the 25 mm EFL lens adds about 76 mm to the overall optical length.

In the lab bench setup the 25 mm EFL collection lens was focused for an image test. Fig. 6 shows that all the beads in the test plate were imaged. The bright spots are the result of room light scattering from the uncoated field lens assembly surfaces. Other test images were examined with better scattering control using photoluminescent test beads and the extreme positions of the beads were observed.

In preliminary tests the position of the field lens assembly was not sensitive to +1-mm movements. This positioning of the field lens assembly was saved for later photoluminescent testing to maximize the energy collected from the extreme bead positions. The collection imaging lens had a fixed Ft# aperture diameter and the separation distance positions were optimized such that the light emitted passed through the fixed aperture.

Discussion of the use of Fresnel lenses for field lenses Fresnel lenses are typically used in applications where efficiency in projecting large bundles of light is desired such as search lights or lighthouses. Non-Fresnel surfaces using spherical or aspherical elements have larger volumes, weigh more and cost more to produce. The optical function of collimating/directing the light bundle in a cylindrical path is better than continuous spherical surfaces. Most spherical and aspherical lenses have uncorrectable aberrations that cause the projected beam to expand over short distances.

The expansion of the projected beam is considered inefficient. The reverse is true with collectors such as solar concentrators. A Fresnel lens will form a more concentrated spot of energy.

For imaging applications Fresnel lenses are used when expense is a manufacturing factor and scattered light and lower resolution is not a concern for the viewers. Most Fresnel lenses are polymer materials which can be cast or injection molded making them less expensive than glass components. In most imaging applications using sensors or small viewing screens the scattered light will be noticeable and cause a loss of contrast.

The manufacturing/molding errors of prismatic surfaces will contribute to the light scattering and uncorrectable aberrations. Theoretical transmission expressions approach 90% for nearly perfect surfaces and very good transitions between one circular prism to its neighbor. Some of the literature shows transmission between 70-90% due to surface or molding errors. In optical ray tracing discussed and illustrated below scattering becomes apparent. The most general comment against using Fresnel lens in systems where some resolution is required, is that the errant deviation of some of the light that is collected by the imaging lens will cause unpredictable spreading of the energy in the image of the test spots. When the energy in the image of the test spots increases in size, the images blur together and become undetectable as single spots.

The use of a Fresnel lens for comparative purposes to the preferred embodiment will now be described in more detail with reference to Figs. 7-10. It should be understood that a Fresnel lens suffers from a number of problems which are discussed in more detail below. An imaging system using a Fresnel field lens is not intended to fall within the scope of the present invention.

Fig. 7 shows a test or sample plate on the lefthand side. A Fresnel lens is shown having the same focal length as previous examples. A collection imaging lens of 25 mm is shown on the righthand side. This example shows only a few rays traced from the test object so that the rays can be seen.

It is apparent from Fig. 7 that some of the light has deviated from the test ray bundles. In actual real use more rays would deviate between these extreme angles and the normal rays directed toward the collection imaging lens. The deviated rays would not be corrected to focus in the same way as other rays. As a result, the deviations would increase the size of the imaged bead.

Fig. 8 shows in greater detail how the transitions from neighboring surfaces causes some light rays to deviate. The optical simulation shown in Fig. 8 is perfect with no errors due to molding or casting. The line between the prisms is the draft angle, typically 1-5°. In the particular example shown in Figs. 8 and 8A the angle is 2°. The draft angle is necessary to allow the polymer surface to be removed from the mold or casting. The larger the draft angle, the greater the number of rays that will show deviation. This example is a typical density of lines or prisms per unit length of 100 lines/inch (4/mm).

As the rays overlap neighboring prisms, the scattering becomes more apparent.

Here the prisms are shown with no errors, so the rays are deviated/reflected at twice the draft angle. With errors on the draft surface, rays will deviate from the ideal direction as shown. This deviation can be smaller than the reflected rays and will be displaced from the ideal focused position leading to less resolution.

In the arrangement shown in Fig. 9 the surface of the Fresnel lens is arranged about 11 mm above from the optical axis. This is where a combination of rays are deviated by the prism and are not deviated by the space between prisms. The undeviated rays are focused 0.02 mm from the deviated prism rays. This deviation is shown in a perfect system and a perfect collection lens. Actual results would show larger deviations with real components. What cannot be shown in the ray tracing examples are all the deviations accumulated from the total surface of the Fresnel lens. This structuring will reduce the contrast in a manner similar to the large deviated rays.

Fig. 10 shows some rays passing through the prism perfectly whereas other rays do not. Fig. 1OA shows the rays being perfectly bent by the prism.

Fig. lOB shows the rays in the sensor or image plane. The spot on the left is from the rays with some deviation. The spot is showing two concentrations of rays. The lower spot shows the deviated rays. If rays could be collected from the portion of the circular surface illuminated by the whole test spot, the lower spot would enlarge and reduce the resolution.

The spot on the right is an example of perfectly directed rays from the prism. The difference in spot sizes is about 2:1 on the RMS calculation which relates to the general resolution.

Fig. bC shows ray tracings from four locations around a 1 mm circle and one center location. The effect of the uncorrectable deviations is shown. Fig. 1OD shows all five spots shown in the sensor plane.

If the system were perfect the size of this spot would be 0.177 mm OD. The size of these spots is approximately 0.3 mm.

The above discussion shows that using a Fresnel lens would result in aberrations and uncorrectable deviations.

Therefore, according to the present invention a Fresnel lens is not used and the present invention does not, therefore, suffer from the problems that would be inherent if a Fresnel lens were to be used.

Field lens configuration ojtimization

An initial study was performed to determine how many field lenses would be needed to minimize the contributed aberrations in conjunction with an off-the-shelf FI0.95 mm EFL (effective focal length) collection lens. The study used off-the-shelf field lenses, modified field lenses, and custom field lenses. A preliminary analysis was done to study the combination of the aberrations of the field lens with a potential custom collection lens, an attempt to balance the aberrations of both.

Fig. 11 shows a low index of a refraction lens with a single powered surface. Fig. 1 1A shows a Modulation Transfer Function ("MTF") chart corresponding to Fig. 11 using a perfect 25 mm EFL collection imaging lens. In Fig. 1 1A and subsequent figures showing a Modulation Transfer Function "T" indicates tangential and "S' indicates sagittal.

The details of interest are the modulation (contrast) values between 4-6 lpfmm (line pairs per mm). This resolution range represents the size of a 1 mm test spot (i.e. bead) inside the test plate well.

Fig. 12 shows two low index of refraction lenses that were tested in lab trials with a mm F/0.95 collection lens. Fig. 12A shows the corresponding Modulation Transfer Function.

Noting the resolution of interest, an improvement is seen by using two powered surfaces over one powered surface.

Fig. 13 shows a single field lens with two powered surfaces. The two radii are from off-the-shelf lenses that can be modified for further lab tests. Fig. 1 3A shows a corresponding Modulation Transfer Function.

The lens shown in Fig. 13 was built for testing with a Ff0.95 collection lens in a cone mechanical configuration for lower weight. Comparing the resolution contrast of Fig. l3Ato Fig. 12A, this approach can be seen to be an improvement and also results in a weight reduction.

Two examples are shown here which optimize different field lens configurations with a true collection lens design.

Fig. 14 shows a field lens configuration optimised with a collection imaging lens.

The embodiment is an example of minimizing the aberrations of the two sets of optical elements. Fig. 14A shows a corresponding Modulation Transfer Function.

With regard to Fig. 14A, it is noted that the curves for different test plate field positions are closer together. Advantageous, an overall improvement for all field positions is observed.

Fig. 15 shows a less preferred embodiment of the present invention wherein two field lenses both having a meniscus shape are used. Various other general approaches to lens shapes may also be used. This approach shows some improvement over the other configuration.

A four element collector lens is shown in Figs. 14 and 15. According to an embodiment the FI# of the collector lens is approx. F/0.9. Other embodiments are contemplated wherein the collector lens has Ff0.8 -according to such embodiments the collector lens assembly preferably comprises more than four elements.

According to the preferred embodiment light is emitted from the reagent beads due to chemiluminescence and is relatively monochromatic with a peak around 460 nm.

However, the light is nonetheless likely to have a spectrum which needs to be accounted for with the optical design and coatings used.

Fig. 16A shows lens elements according to a particularly preferred embodiment and Fig. 16B shows optical paths through the preferred lens elements.

According to the preferred embodiment the lens elements may comprise a negative meniscus field lens 1 and a collection lens assembly comprising a piano-convex lens 2, a biconvex lens 3, a biconcave lens 4, a piano-concave or bi-concave lens 5, a biconvex lens 6 and a negative meniscus lens 7.

The field lens 1 preferably has a radius of curvature of 129 mm (on the first convex side hereinafter "CX") and 318 mm (on the second concave side hereinafter "CC"), the piano-convex lens 2 preferably has a radius of curvature of 60 mm CX, the biconvex lens 3 preferably has a radius of curvature of 32 mm CX and 32 mm CX, the biconcave lens 4 preferably has a radius of curvature of 32 mm CC and 23 mm CC, the piano-concave or biconcave lens 5 preferably has a radius of curvature of 175mm CC (and 31 mm CC if biconcave), the biconvex lens 6 preferably has a radius of curvature of 31 mm CX and 31 mm CX and the negative meniscus lens 7 preferably has a radius of curvature of 24 mm CX and 36 mm CC.

Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.

Claims (4)

1. <claim-text>Claims 1. An imaging system for imaging a plurality of reagent beads retained in a sample well of a sample plate, comprising:a non-Fresnel field lens or field lens assembly;a collection lens assembly; and a camera sensor array; wherein said collection lens assembly is positioned between said non-Fresnel field lens or field lens assembly and said camera sensor array.</claim-text> <claim-text>2. An imaging system as claimed in claim 1, wherein said field lens or field lens assembly comprises a negative meniscus lens.</claim-text> <claim-text>3. An imaging system as claimed in claim 1 or 2, wherein said collection lens assembly comprises a first lens.</claim-text> <claim-text>4. An imaging system as claimed in claim 3, wherein said first lens comprises a pIano-convex lens.</claim-text> <claim-text>5. An imaging system as claimed in claim 3 or4, wherein said collection lens assembly comprises a second lens and wherein light from said reagent beads passes, in use, from said first lens to said second lens.</claim-text> <claim-text>6. An imaging system as claimed in claim 5, wherein said second lens comprises a biconvex lens.</claim-text> <claim-text>7. An imaging system as claimed in any of claims 3-6, wherein said collection lens assembly comprises a third lens and wherein light from said reagent beads passes, in use, from said second lens to said third lens.</claim-text> <claim-text>8. An imaging system as claimed in claim 7, wherein said third lens comprises a biconcave lens.</claim-text> <claim-text>9. An imaging system as claimed in any of claims 3-8, wherein said collection lens assembly comprises a fourth lens and wherein light from said reagent beads passes, in use, from said third lens to said fourth lens.</claim-text> <claim-text>10. An imaging system as claimed in claim 9, wherein said fourth lens comprises a piano-concave or biconcave lens.</claim-text> <claim-text>-20 - 11. An imaging system as claimed in any of claims 3-10, wherein said collection lens assembly comprises a fifth lens and wherein light from said reagent beads passes, in use, from said fourth lens to said fifth lens.</claim-text> <claim-text>12. An imaging system as claimed in claim 11, wherein said fifth lens comprises a biconvex lens.</claim-text> <claim-text>13. An imaging system as claimed in any of claims 3-12, wherein said collection lens assembly comprises a sixth lens and wherein light from said reagent beads passes, in use, from said fifth lens to said sixth lens.</claim-text> <claim-text>14. An imaging system as claimed in claim 13, wherein said sixth lens comprises a negative meniscus lens.</claim-text> <claim-text>15. An imaging system as claimed in any preceding claim, wherein said collection lens assembly has a F/# selected from the group consisting of: (i) F/0.70 -FI0.80; (U) F/0.80 -Ff0.90; (Ui) FJ0.90 -F/i.00; and (iv) F/i.00 -Ff1.10.</claim-text> <claim-text>16. An imaging system as claimed in any preceding claim, wherein said non-Fresnel field lens or field lens assembly and/or said collection lens assembly has a magnification selected from the group consisting of: (i) <0.12; (ii) 0.12-0.14; (iii) 0.14-0.16; (iv) 0.16-0.18; (v) 0.18-0.20; and (vi) > 0.20.</claim-text> <claim-text>17. An imaging system as claimed in any preceding claim, wherein an optical path length from a base portion of said sample well of said sample plate to the detecting surface of said camera sensor array is selected from the group consisting of: (i) 50-i 00 mm; (ii) 100-1 50 mm; (iii) 150-200 mm; (iv) 200-250 mm; (v) 250-300 mm; (vi) 300-350 mm; (vU) 350-400 mm; and (vUi) 400-450 nm; (ix) 450-500 nm.</claim-text> <claim-text>18. An imaging system as claimed in any preceding claim, wherein said camera sensor array comprises a charge coupled device (CCD) camera.</claim-text> <claim-text>19. A macroarrayer comprising an imaging system as claimed in any preceding claim.</claim-text> <claim-text>20. A macroarrayeras claimed in claim 19, further comprising a sample plate.</claim-text> <claim-text>21. A macroarrayer as claimed in claim 20, wherein said sample well of said sample plate has an internal diameter selected from the group consisting of: (i) 5-6 mm; (U) 6-7 mm; (iii) 7-8 mm; (iv) 8-9 mm; (v)9-10 mm; (vi) 10-il mm; (vU) 11-12 mm; (viii) 12-13 mm; (ix) 13-14 mm; (x) 14-15 mm; (xi) 15-16 mm; (xii) 16-17 mm; (xiU) 17-18 mm; (xiv) 18-19 mm; and (xv) 19-20 mm. -21 -</claim-text> <claim-text>22. A macroarrayer as claimed in claim 20 or 21, wherein said reagent beads have a diameter selected from the group consisting of: (I) C 0.5 mm; (ii) 0.5-1.0 mm; (iii) 1.0-1.5 mm; (iv) 1.5-2.0 mm; (v)2.0-2.5 mm; (vi)
2. 2.5-3.0 mm; (vii)
3. 3.0-3.5 mm; (viii) 3.5-4.0 mm; (ix)
4. 4.0-4.5 mm; and (x) 4.5-5.0 mm.</claim-text> <claim-text>23. A macroarrayer as claimed in claim 20, 21 or 22, wherein said sample well has a base portion having a plurality of blind recesses or through-holes, wherein a reagent bead is inserted or retained in each of said blind recesses or through-holes and wherein the depth of said sample well as measured from an opening into said sample well through which sample is dispensed in use to said base portion is selected from the group consisting of: (02-3 mm; (U)3-4 mm; (iii) 4-5 mm; (iv) 5-6 mm; (v) 6-7 mm; (vi)7-8 mm; (vU) 8-9 mm; and (vHi) 9-10 mm.</claim-text> <claim-text>24. A macroarrayer as claimed in any of claims 20-23, further comprising a processing system, said processing system being arranged and adapted to detect and quantify luminescence of reagent beads in said sample plate as an indicator of the presence or amount of a target compound.</claim-text> <claim-text>25. A macroarrayer as claimed in any of claims 20-24, wherein light is emitted, in use, from said reagent beads due to bioluminescence or chemiluminescence and has a peak in a range selected from the group consisting of: (i) 400-410 nm; (ii) 410-420 nm; (iii) 420-430 nm; (iv) 430-440 nm; (v) 440-450 nm; (vi) 450-460 nm; (vU) 460-470 nm; (viU) 470-480 nm; (ix) 480-490 nm; and (x) 490-500 nm.</claim-text> <claim-text>26. A luminometer for analysing a plurality of luminescent samples comprising:a non-Fresnel field lens or field lens assembly;a collection lens assembly; and a camera sensor array; said collection lens assembly being positioned between said non-Fresnel field lens or field lens assembly and said camera sensor array.</claim-text> <claim-text>27. A luminometer as claimed in claim 26, wherein said plurality of luminescent samples comprise bioluminescent or chemiluminescent samples.</claim-text> <claim-text>28. A method of imaging a plurality of reagent beads retained in a sample well of a sample plate, comprising: providing a non-Fresnel field lens or field lens assembly, a collection lens assembly and a camera sensor array, wherein said collection lens assembly is positioned between said non-Fresnel field lens or field lens assembly and said camera sensor array; and using said camera sensor array to determiner the intensity of said reagent beads.</claim-text> <claim-text>-22 - 29. An imaging system for imaging a plurality of reagent beads retained in a sample well of a sample plate, comprising: a non-Fresnel field lens or field lens assembly comprising a negative meniscus lens; a collection lens assembly, said collection lens assembly comprising in order a first lens comprising a piano-convex lens, a second lens comprising a biconvex lens, a third lens comprising a biconcave lens, a fourth lens comprising a piano-concave or biconcave lens, a fifth lens comprising a biconvex lens and a sixth lens comprising a negative meniscus lens; and a camera sensor array; wherein said collection lens assembly is positioned between said non-Fresnel field lens or lens assembly and said camera sensor array.</claim-text> <claim-text>30. A method of imaging a plurality of reagent beads retained in a sample well of a sample plate, comprising: providing a non-Fresnel field lens or lens assembly comprising a negative meniscus lens; providing a collection lens assembly, said collection lens assembly comprising in order a first lens comprising a piano-convex lens, a second lens comprising a biconvex lens, a third lens comprising a biconcave lens, a fourth lens comprising a piano-concave or biconcave lens, a fifth lens comprising a biconvex lens and a sixth lens comprising a negative meniscus lens; providing a camera sensor array, wherein said collection lens assembly is positioned between said non-Fresnel field lens or lens assembly and said camera sensor array; and using said camera sensor array to determine the intensity of said reagent beads.</claim-text>