WO2025117598A1

WO2025117598A1 - Illumination system for a bio-analytical instrument

Info

Publication number: WO2025117598A1
Application number: PCT/US2024/057549
Authority: WO
Inventors: Mingsong Chen
Original assignee: Life Technologies Holdings Pty Ltd
Current assignee: Life Technologies Holdings Pty Ltd
Priority date: 2023-11-28
Filing date: 2024-11-26
Publication date: 2025-06-05
Anticipated expiration: 2026-05-28

Abstract

Disclosed are illumination systems, and bioanalytical instruments comprising the illumination systems. The illumination system comprises two or more staggered mirrors configured to split an incoming light beam into at least two sub-beams, and a guiding module configured to direct the sub-beams to provide oblique illumination to an area. The illumination system is capable of improving illumination efficiency, reducing background signal, and improving image uniformity compared to conventional illumination systems used for bioanalytical instruments.

Description

ILLUMINATION SYSTEM FOR A BIO-ANALYTICAL INSTRUMENT

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/603,345 filed November 28, 2023, which is incorporated herein by reference.

FILED OF INVENTION

[0002] The present disclosure concerns optical systems. More specifically, the disclosure is related to an illumination system for a bio-analytical instrument. Even more specifically, the present disclosure relates to an illumination system that includes staggered minors for splitting source light and providing oblique illumination to biological samples.

BACKGROUND OF THE INVENTION

[0003] Conventional illumination systems for bio-analytical instruments are generally configured to provide on-axis illumination for biological samples, where the excitation light beams and/or illumination light beams and the emission from samples are on the same axis. In these systems, either dichroic beam splitters or a 50/50 beam splitter is used to reflect illumination light and/or excitation light to the samples and to let emission light pass through toward the optical detector or camera. However, on-axis illumination usually causes more light reflected and scattered from sample holders to enter the optical detector or camera, resulting in high background signal for data acquisition and/or image capturing for the biological samples.

[0004] Additionally, when a single 50/50 beam splitter is used for an optical system, at least 75% optical power (watt) is lost, which could significantly limit the data accuracy when it is used to provide illumination for samples with low sample volumes, low concentrations, and/or low emission intensity. On the other hand, although dichroic beam splitters have high optical efficiency, each optical channel is coupled with a dichroic beam splitter, thereby drastically increasing footprint and the overall cost of the bio-analytical instrument, which generally has multiple optical channels. Moreover, existing optical systems for bio-analytical instruments indiscriminately provide illumination to both sample-containing areas and non-sample containing areas of a sample holder/sample plate, which can lead to wasted optical power (watt) and high background signal reflected and scattered by the non-sample containing areas.

[0005] Overall, while illumination systems for bio-analytical instrument exist, the need for improvements in this field persists in light of at least the aforementioned drawbacks for these illumination systems.

SUMMARY OF THE INVENTION

[0006] A solution to at least some of the above-mentioned problems associated with illumination systems of bio-analytical instruments has been discovered. The solution resides in an illumination system that comprises staggered mirrors that are configured to split a source light beam into multiple sub-beams, which are subsequently directed to sample containing areas of a sample holder. This can be beneficial for avoiding illumination to non- sample containing areas of sample holder, thereby reducing energy consumption and footprint for the light source.

[0007] Additionally, by avoiding illumination to non-sample areas of sample holders, the staggered mirrors can significantly improve the illumination efficiency, which allows the illumination system disclosed herein to be used for illuminating small sample volumes, low sample concentrations, and/or low emission intensities. By way of example, the illumination system of the present disclosure can be used for a digital PCR assay, which requires high illumination efficiency as each reaction site and/or sample partition of the sample plate contains low concentration and/or low volume of sample/probe.

[0008] Furthermore, the illumination system comprises a guiding module for directing the sub-beams from the staggered mirrors to provide oblique illumination to biological samples. Notably, implementing oblique illumination can lower background signal compared to existing illumination systems for on-axis illumination, resulting in higher image and/or data quality when the sample images are taken.

[0009] Still further, the staggered mirrors of the illumination system disclosed herein can be configured to direct inner portions of a source light beam to provide illumination to outer portions of sample plates, such that the outer portions of the sample plate receive higher light intensity than the inner portions. This can mitigate the vignetting and/or roll-off effect for the images of the sample plate, resulting in higher image uniformity and improved image-based data quality. Therefore, the illumination systems disclosed herein provide a technical achievement over at least some of the problems associated with the currently available illumination systems mentioned above.

[0010] Certain embodiments are directed to illumination systems. In certain aspects, the illumination system comprises two or more staggered mirrors that are configured to split an incoming light beam into at least two sub-beams. The illumination system comprises a guiding module configured to direct the sub-beams to provide oblique illumination to an area. The area can include a sample plate for biological samples.

[0011] Certain embodiments are directed to illumination systems. In certain aspects, the illumination system comprises n staggered mirrors configured to split an incoming light beam into m sub-beams. The illumination system comprises a guiding module configured to direct the subbeams to provide oblique illumination to a sample plate. The sample plate comprises k zones. Each zone comprising a plurality of reaction sites, n, m, and k, are each an integer, and m > k, 2<m<n.

[0012] Certain embodiments are directed to instruments for biological analysis. In certain aspects, the instrument comprises a base configured to receive one or more sample plates containing biological samples. The instrument comprises an illumination system configured to illuminate the one or more sample plates. The illumination system comprises n staggered mirrors configured to split an incoming light beam into m sub-beams. The illumination system comprises one or more front mirrors configured to direct the sub-beams to provide oblique illumination to the one or more sample plates and/or to excite the biological samples to produce emission light. In some instances, the incoming light beam and the sub-beams are substantially collimated or collimated light beams. The sample plate comprises k zones, each zone comprises a plurality of reaction sites, where n, m, and k, are each an integer, and m > k, 2<m<n. The instrument comprises an optical sensor configured to receive the emission light from the biological samples. [0013] The following includes definitions of various terms and phrases used throughout this specification.

[0014] The terms “about” or “approximately” are defined as being close to as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%, preferably, within 5%, more preferably, within 1%, and most preferably, within 0.5%.

[0015] The term “substantially” and its variations are defined to include ranges within 10%, within 5%, within 1%, or within 0.5%.

[0016] As used herein, an "emission", or “emission light” means an electromagnetic radiation produced as the result an interaction of radiation from an excitation source (e.g, an excitation light) with one or more samples containing, or thought to contain, one or more chemical and/or biological molecules or compounds of interest. The emission may be due to a reflection, refraction, polarization, absorption, and/or other optical effect by a sample on radiation from an excitation source. For example, the emission may comprise a luminescence or fluorescence induced by absorption of the excitation electromagnetic radiation (e.g., excitation light) by one or more samples.

[0017] As used herein, a lens means an optical element configured to direct or focus incident electromagnetic radiation so as to converge or diverge such radiation, for example, to provide a real or virtual image, either at a finite distance or at an optical infinity. The lens may comprise a single optical element having a focusing power provided by refraction, reflection, and/or diffraction of the incident electromagnetic radiation. Alternatively, the lens may comprise a compound system including a plurality of optical element, for example, including, but not limited to, an achromatic lens, doublet lens, triplet lens, or camera lens. The lens may be at least partially housed in or at least partially enclosed by a lens case or a lens mount.

[0018] As used herein, the term "biological sample" means a sample or solution containing any type of biological chemical or component and/or any target molecule of interest to a user, manufacturer, or distributor of the various embodiments of the present invention described or implied herein, as well as any sample or solution containing related chemicals or compounds used for the purpose of conducting a biological assay, experiment, or test. These biological chemicals, components, or target molecules may include, but are not limited to, DNA sequences (including cell-free DNA), RNA sequences, genes, oligonucleotides, molecules, proteins, biomarkers, cells (e.g., circulating tumor cells), or any other suitable target biomolecule. A biological sample may comprise one or more of at least one target nucleic acid sequence, at least one primer, at least one buffer, at least one nucleotide, at least one enzyme, at least one detergent, at least one blocking agent, or at least one dye, marker, and/or probe suitable for detecting a target or reference nucleic acid sequence. In various embodiments, such biological components may be used in conjunction with one or more PCR methods and systems in applications such as fetal diagnostics, multiplex dPCR, viral detection, and quantification standards, genotyping, sequencing assays, experiments, or protocols, sequencing validation, mutation detection, detection of genetically modified organisms, rare allele detection, and/or copy number variation.

[0019] The term “beam”, “light beam”, and their variations are defined to include a directional projection of light energy radiating from a light source.

[0020] The term “staggered mirrors” and its variations are defined to include mirrors that are offset from each other in at least one direction.

[0021] The term “collimate” and its variations disclosed throughout the specification are defined as to make rays of a light beam or particles substantially accurately parallel with a maximum angle of 3 degree, or 5 degree, or more depending on specific requirements including illumination uniformity or image uniformity requirements. In certain aspects, one or more collimating lenses can be used to collimate a light beam.

[0022] The term “oblique illumination”, “off-axis illumination” and their variations disclosed throughout the specification are defined as illumination light strikes samples or specimen at an oblique angle rather than perpendicularly to the samples or specimen.

[0023] The term “illumination efficiency” and its variations disclosed throughout the specification are defined as the ratio of the optical power (watt) received by sample containing areas of a sample holder or sample plate to the total optical power (watt) from the light source of the illumination system. [0024] The terms “inhibiting” or “reducing” or “preventing” or “avoiding” or any variation of these terms, when used in the claims and/or the specification, includes any measurable decrease or complete inhibition to achieve a desired result.

[0025] The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.

[0026] The use of the words “a” or “an” when used in conjunction with the term “comprising,” “including,” “containing,” or “having” in the claims or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0027] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0028] The system and/or instrument of the present disclosure can “comprise,” “consist essentially of,” or “consist of’ particular components, compositions, etc., disclosed throughout the specification. With respect to the transitional phase “consisting essentially of,” in one non-limiting aspect, a basic and novel characteristic of the illumination system disclosed in the specification is the staggered mirrors configured to split the incoming beam into sub-beams, and guiding the subbeams to provide oblique illumination to an area.

[0029] Other objects, features and advantages of the embodiments will become apparent from the following figures, detailed description, and examples. It should be understood, however, that the figures, detailed description, and examples, while indicating specific embodiments, are given by way of illustration only and are not meant to be limiting. Additionally, it is contemplated that changes and modifications within the spirit and scope will become apparent to those skilled in the art from this detailed description. In further embodiments, features from specific embodiments may be combined with features from other embodiments. For example, features from one embodiment may be combined with features from any of the other embodiments. In further embodiments, additional features may be added to the specific embodiments described herein.

DESCRIPTION OF THE DRAWINGS

[0030] For a more complete understanding, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0031] FIG. 1A illustrates an illumination system comprising two collimating lenses, according to embodiments disclosed in the specification;

[0032] FIG. IB illustrates an illumination system comprising a single collimating lens, according to embodiments disclosed in the specification;

[0033] FIGS. 2A and 2B illustrate two different configurations of staggered mirrors that direct different portions of the incoming light beam for providing illumination to the inner portions and outer portions of a sample plate, according to embodiments disclosed in the specification; FIG. 2A shows a configuration of staggered mirrors that is used to direct the outer portions of the incoming light beam to the inner portions of a sample plate and inner portions of the incoming light beam to the outer portions of the sample plate. FIG. 2B shows a configuration of staggered mirrors that is used to direct the outer portions of the incoming light beam to the outer portions of the a sample plate and inner portions of the incoming light beam to the outer portions of the sample plate.

[0034] FIG. 3A-3D illustrate sample plate images captured using the illumination system disclosed in the specification. FIG. 3A shows an image taken for a FAM™ dye loaded sample plate with staggered mirrors in configuration shown in FIG. 2B; FIG. 3B shows an image taken for a FAM™ dye loaded sample plate with staggered mirrors in configuration of FIG. 2A; FIG. 3C shows an image taken for a VIC® dye loaded sample plate with staggered mirrors in configuration of FIG. 2B; FIG. 3D shows an image taken for a VIC dye loaded sample plate with staggered mirrors in configuration of FIG. 2A. DETAILED DESCRIPTION OF THE INVENTION

[0035] The currently available illumination systems for bioanalytical instruments suffer several deficiencies including low illumination efficiency, high background signal, large footprint, and/or high manufacturing costs. The embodiments of the present invention provide a solution to at least some of these problems. The solution is premised on an illumination system comprising two or more staggered mirrors configured to split an incoming light beam into multiple sub-beams and direct the sub-beams to sample containing areas of a sample holder, thereby improving illumination efficiency and reducing required power and size of a light source of the illumination system. Furthermore, the illumination system disclosed herein comprises a guiding module configured to guide the sub-beams from the staggered mirrors to provide oblique illumination to sample plates, resulting in reduced background signal. Moreover, the staggered mirrors of the illumination system can be configured to mitigate vignetting and/or roll-off effect of sample images, which can lead to improved image uniformity and data quality for the bio-analytical instruments.

[0036] These and other non-limiting aspects of the present invention are discussed in further detail in the following sections.

A. Illumination System

[0037] Illumination systems for bio-analytical instruments are generally designed to provide illumination for imaging and/or excitation of biological samples. For conventional illumination systems of bio-analytical instruments, particularly polymerase chain reaction (PCR) instruments, on-axis illumination is used since sample/probe concentrations, sample volumes and inherently emission intensity for conventional PCR or qPCR are sufficient to overcome low illumination efficiency and high background signals associated with on-axis illumination. However, when sample concentrations, sample volumes and/or emission intensity are relatively low, for instance in a digital PCR assay, the currently available illumination systems can be disadvantageous in terms of image and/or data quality. The illumination systems disclosed in the specification implement oblique illumination (off-axis) to the samples, thereby improving image uniformity and/or data quality for biological assays. [0038] With reference to FIGS. 1A, a schematic diagram is shown for illumination system 100, which can be used for providing illumination and/or excitation for biological samples. According to embodiments, illumination system 100 comprises light source 101 configured to provide source light beam 11. Light source 101 can be in any shape including round shape, square shape, rectangular shape, triangle shape, sector shape, an irregular shape, or any combination thereof. In some embodiments, light source 101 comprises a light emitting diode (LED) light source, an organic LED (OLED) light source, a tungsten halogen light source, a laser light source, a xenon light source, an argon light source, a krypton light source, an incandescent light source, any other type of commercially available light source, or a combination thereof. Light source 101 may have a power of 6 to 8 watt at the specific driving current and all ranges or values therebetween including ranges of 6 to 6.2 watt, 6.2 to 6.4 watt, 6.4 to 6.6 watt, 6.6 to 6.8 watt, 6.8 to 7.0 watt, 7.0 to 7.2 watt, 7.2 to 7.4 watt, 7.4 to 7.6 watt, 7.6 to 7.8 watt, and 7.8 to 8.0 watt. Light source 101 may have a bro ad- spectrum range as required by the bio-analytical instrument. Light source 101 may have a balanced optical power (watt) distribution across the spectrum. In some embodiments, source light beam 11 is a divergent light beam.

[0039] In some embodiments, illumination system 100 comprises beam shaper 102 configured to narrow down and/or shape source light beam 11 to produce narrowed light beam 12. Illumination system 100 can comprise collimator 103 configured to collimate narrowed light beam 12 to produce incoming light beam 13. Incoming light beam 13 is a collimated or substantially collimated light beam. In some embodiments, collimator 103 of illumination system 100 comprises two cylindrical collimating lenses (103a and 103b) with different focal lengths configured to generate a rectangular incoming light beam 13. In some embodiments, the two cylindrical collimating lenses 103a and 103b are both rectangular. In some aspects, collimating lens 103a has a focal length of 350 to 450 mm and all ranges and values there between including ranges of 350 to 360 mm, 360 to 370 mm, 370 to 380 mm, 380 to 390 mm, 390 to 400mm, 400 to 410 mm, 410 to 420 mm, 420 to 430 mm, 430 to 440 mm, and 440 to 450 mm. Collimating lens 103b has a focal length of 450 to 550 mm and all ranges and values there between including ranges of 450 to 460 mm, 460 to 470 mm, 470 to 480 mm, 480 to 490 mm, 490 to 500mm, 500 to 510 mm, 510 to 520 mm, 520 to 530 mm, 530 to 540 mm, and 540 to 550 mm. Rectangular collimating lens 103a and collimating lens 103b are positioned with different directions such that incoming light beam is shaped into a rectangular shape. By way of example, in some embodiments, rectangular collimating lens 103a is positioned such that the convex edges in the vertical direction, and rectangular collimating lens 103b is positioned such that the convex edges are in the horizontal direction. Collimating lenses 103a and 103b are made of borosilicate glass (e.g. Schott BK7®), plastic, a combination of concave lens of flint glass with convex lens of crown glass as achromatic doublet, or a combination thereof. In some aspects, collimating lenses 103a and 103b have a wavelength range of 400 to 800 nm. According to embodiments, a light path from light source 101 to collimator 103 is in a range of 350 mm to 550 mm and all ranges and values there between including ranges of 350 mm to 360 mm, 360 mm to 370 mm, 370 mm to 380 mm, 380 mm to 390 mm, 390 mm to 400 mm, 400 mm to 410 mm, 410 mm to 420 mm, 420 mm to 430 mm, 430 mm to 440 mm, 440 mm to 450 mm, 450 mm to 460 mm, 460 mm to 470 mm, 470 mm to 480 mm, 480 mm to 490 mm, 490 mm to 500 mm, 500 mm to 510 mm, 510 mm to 520 mm, 520 mm to 530 mm, 530 mm to 540 mm, and 540 mm to 550 mm.

[0040] In some embodiments, illumination system 100 comprises staggered mirrors 104 (e.g., 104a, 104b, 104c, and 104d as shown in FIG.1A) configured to split incoming beam 13 into a plurality sub-beams 14 (e.g. 14a, 14b, 14c, and 14d). The splitting by staggered mirrors 104 includes that each of staggered mirrors reflects a portion of incoming light beam 13 such that each reflected portion of incoming light beam 13 forms a sub-beam (e.g., 14a, 14b, 14c, or 14d). According to embodiments disclosed in the specification, staggered mirrors 104 are configured such that there is substantially no loss of light when incoming light beam 13 is split into sub-beams 14. Sub-beams 14 may be substantially parallel to each other. Each of sub-beams 14 is a collimated or substantially collimated beam.

[0041] Staggered mirrors 104 comprise optical mirrors. The optical mirrors has an reflectance for reflecting light. Staggered minors 104 (e.g., 104a, 104b, 104c, and 104d) are not in the same plane. Staggered mirrors 104 (e.g., 104a, 104b, 104c, and 104d) may be substantially parallel to each other. In some aspects, staggered mirrors 104 are positioned such that incoming light beam 13 has an angle of incidence to staggered mirrors 104 of 30 to 60 degrees and all values and ranges there between including ranges of 30 to 32 degrees, 32 to 34 degrees, 34 to 36 degrees, 36 to 38 degrees, 38 to 40 degrees, 40 to 42 degrees, 42 to 44 degrees, 44 to 46 degrees, 46 to 48 degrees, 48 to 50 degrees, 50 to 52 degrees, 52 to 54 degrees, 54 to 56 degrees, 56 to 58 degrees, and 58 to 60 degrees. In some instances, the angle of incidence for incoming beam 13 to staggered mirrors 104 is 45 degrees.

[0042] According to embodiments disclosed in this specification, illumination system 100 comprises guiding module 105 configured to direct sub-beams 14 (e.g., 14a-14d) to provide oblique illumination to the area. In some instances, guiding module 105 comprises one or more front mirrors configured to reflect sub-beams 14 (e.g., 14a-14d) to produce reflected sub-beams 15 (e.g., 15a-15d) such that reflected sub-beams 15 (e.g., 15a-15d) provide oblique illumination to the area. Reflected sub-beams 15 (e.g., 15a-15d) are collimated or substantially collimated light beams according to some embodiments. In some aspects, an angle of incidence of sub-beams 14 (e g., 14a-14d) to the front mirror(s) is in a range of 20 to 40 degrees and all ranges and values there between including ranges of 20 to 22 degrees, 22 to 24 degrees, 24 to 26 degrees, 26 to 28 degrees, 28 to 30 degrees, 30 to 32 degrees, 32 to 34 degrees, 34 to 36 degrees, 36 to 38 degrees, and 38 to 40 degrees. In some instances, guiding module 105 comprises a single front mirror and the angle of incidence of sub-beams 14 to the front mirror is 32 degrees. In some other instances, guiding module 105 comprises a plurality of front mirrors, and the angle of incidence of sub-beams 14 (e.g., 14a-14d) to the front mirrors is 20-40 degrees. Reflected sub-beams 15 has an angle of incidence to the area of 20 to 40 degrees and all ranges and values there between including ranges of 20 to 22 degrees, 22 to 24 degrees, 24 to 26 degrees, 26 to 28 degrees, 28 to 30 degrees, 30 to 32 degrees, 32 to 34 degrees, 34 to 36 degrees, 36 to 38 degrees, and 38 to 40 degrees. In some embodiments, the light path from staggered mirrors 104 to the one or more front mirrors of guiding module 105 is from 200 to 400 mm and all ranges and values there between and all ranges and values there between including ranges of 200 to 220 mm, 220 to 240 mm, 240 to 260 mm, 260 to 280 mm, 280 to 300 mm, 300 to 320 mm, 320 to 340 mm, 340 to 360 mm, 360 to 380 mm, and 380 to 400 mm.

[0043] In some embodiments, the area, to which reflected sub-beams 15 (e.g., 15a-15d) provide illumination, includes sample plate 106. Sample plate 106 can comprises a plurality of zones. Each zone of sample plate 106 comprises a plurality of reaction sites. In embodiments, any of two adjacent zones of sample plate 106 were separated by a divider. Each of the dividers receives significantly less illumination directly from reflected sub-beams 15 (e.g. reflected subbeams 15a-15d). In some instances, each of the dividers receives less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 1% illumination directly from reflected sub-beams (e.g., reflected sub-beams 15a-15d) compared to each zone of sample plate. In some embodiments, each of the dividers receives substantially no illumination directly from any reflected sub-beam 15 (e.g., reflected sub-beams 15a-15d). According to embodiments, a light path from light source 101 to sample plate 106 is from 600 mm to 1200 mm and all values and ranges there between including ranges of 600 mm to 650 mm, 650 mm to 700 mm, 700 to 750 mm, 750 mm to 800 mm, 800 to 850 mm, 850 to 900 mm, 900 to 950 mm, 950 to 1000 mm, 1000 to 1050 mm, 1050 mm to 1100 mm, 1100 mm to 1150 mm, and 1150 to 1200 mm. In some instances, the light path from light source 101 to sample plate 106 is about 900 mm.

[0044] In some embodiments, illumination system 100 comprises n staggered mirrors, and incoming light beam 13 is split into m sub-beams 14, and sample plate 106 comprises k zones, where n, m, and k, are each an integer, and m > k, 2<m<n. In some aspects, one or more of subbeams are configured to provide oblique illumination to a single zone of sample plate 106. Each zone of sample plate 106 contains a plurality of reaction sites. In some instances, staggered mirrors 101 are configured such that each of sub-beams 14 (e.g., 14a-14d) provides illumination to one zone of sample plate.

[0045] According to embodiments, staggered mirrors 104 are positioned such that mirrors receiving inner portions of incoming light beam 13 direct corresponding sub-beams 14 to the zones disposed at the outer portions of sample plate 106 and mirrors receiving outer portions of incoming light beam 13 direct corresponding sub-beams 14 (e.g., 14a-14d) to the inner portions of sample plate 106, thereby mitigating vignetting and/or roll-off effect of an imaging lens when images are captured for sample plate 106. In some embodiments, staggered mirrors 104 are not in the same plane. The positioning of staggered mirrors 104, including the distance between the staggered mirrors 104 are determined by the positioning of the zones of sample plate 106.

[0046] In some embodiments, staggered mirrors 101 each can be in any shape including round, rectangular, square, sector, triangular, any irregular shape, or a combination thereof. In some aspects, reflective surfaces of staggered mirrors 104 (e.g., 104a-104d) are each in substantially the same shape as each zone of sample plate 106. Reflective surfaces of staggered mirrors 104 (e g., 104a-104d) each may have substantially the same or larger dimensions than each zone of sample plate 106. In some embodiments, sample plate 106 is disposed underneath manifold 107. Manifold 107 is configured to secure sample plate 106 by force and/or weight. In some aspects, sample plate 106 comprises a plurality of valves disposed along the edges and/or dividers thereof, and manifold 107 is in fluid communication with the valves. Manifold 107 does not cover any of the zones containing reaction sites of sample plate 106. According to some embodiments, the angle of incidence for reflected sub-beams 15 (e.g., 15a-15d) is determined to avoid or minimize shadow of manifold 107 on the zones of sample plate 106. Manifold 107 may be made of plastic, a metal including aluminum and steel, or a combination thereof.

[0047] In some embodiments, with reference to FIG. 2A, sample plate 106 is substantially rectangular. Sample plate 106 can comprise four rectangular zones 301-304. Zone 301 and zone 304 are at the outer portions of sample plate 106. Zone 302 and zone 303 are at the inner portions of sample plate 106. Illumination system 100 may comprise staggered mirrors 104a-104d, configured to split incoming light beam 13 into sub-beams 14a-14d. Each of sub-beams 14a-14d provides oblique illumination to each of zones 301-304 after being reflected by guiding module 105 (not shown). According to some embodiments, as shown in FIG. 2A, sub-beam 14a and subbeam 14d generated by staggered mirrors 104a and 104d reflecting outer portions of incoming light beam 13 are directed to zone 302 and zone 303, which are disposed at the inner portions of sample plate 106. Sub-beam 14b and sub-beam 14c generated by staggered mirrors 104b and 104c reflecting inner portions of incoming light beam 13 are direct to zone 301 and zone 304, which are disposed at the outer portions of sample plate 106. Staggered mirrors 104a-104d are configured to mitigate vignetting and/or roll-off effect of images of sample plate 106, thereby improving image uniformity of sample plate 106.

[0048] According to some embodiments, each zone of sample plate 106 comprises a plurality of reaction sites. Each reaction site can comprise a reaction mixture containing a biological sample. At least some of the reaction sites contain a dye capable of being excited by reflected sub-beams 15 (e.g., 15a-15d). In some embodiments, the biological sample comprise a nucleic acid sample. The nucleic acid sample can include ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA). In some embodiments, sample plate 106 comprises a microfluidic chip. The microfluidic chip comprises a plurality of microchambers. In some embodiments, the microfluidic chip comprises a plurality of through-holes. In some instances, the reaction sites are each contained in a microchamber and/or through -hole of sample plate 106.

[0049] With reference to FIG. IB, as an alternative to collimator 103 that comprises two cylindrical collimating lenses 103a and 103b in illumination system 100, collimator 103 in system 200 as shown in FIG. IB can include a single collimating lens. The single collimating lens has a focal length of 350 to 600 mm and all ranges and values therebetween including ranges of 350 to 375 mm, 375 to 400 mm, 400 to 425 mm, 425 to 450 mm, 450 to 475 mm, 475 to 500 mm, 500 to 525 mm, 525 to 550 mm, 550 to 575 mm, and 575 to 600 mm. The single collimating lens can have a lens diameter of 100 to 130 mm and all ranges and values therebetween including ranges of 100 to 102 mm, 102 to 104 mm, 104 to 106 mm, 106 to 108 mm, 108 to 110 mm, 110 to 112 mm, 112 to 114 mm, 114 to 116 mm, 116 to 118 mm, 118 to 120 mm, 120 to 122 mm, 122 to 124 mm, 124 to 126 mm, 126 to 128 mm, 128 to 130 mm. In some embodiments, the single collimating lens has a wavelength range of 400 to 800 nm. In some aspects, the single collimating lens is made of a material comprising borosilica, plastic, or a combination thereof. As shown in FIG. IB, illumination system 200 includes all the components and features of illumination system 100 except a different configuration of collimator 103.

[0050] According to embodiments, optical efficiency for illumination systems 100 and 200 each have optical efficiency of about 15% to 35% and all ranges and values there between including ranges of 15 to 17%, 17% to 19%, 19% to 21%, 21% to 23%, 23% to 25%, 25% to 27%, 27% to 29%, 29% to 31%, 31% to 33%, and 33% to 35%.

[0051] In some embodiments, illumination systems 100 and 200 are configured for a bio- analytical instrument. Illumination systems 100 and 200 are configured for a polymerase chain reaction (PCR) assay. In some aspects, illumination systems 100 and 200 are used for a digital PCR (dPCR) assay. In a dPCR assay or experiment, according to some embodiments, a dilute solution containing at least one target polynucleotide or nucleotide sequence is subdivided into a plurality of reaction sites, such that at least some of these reaction sites contain either one molecule of the target nucleotide sequence or none of the target nucleotide sequence. When the reaction sites are subsequently thermally cycled in a PCR protocol, procedure, assay, process, or experiment, the reaction sites containing the one or more molecules of the target nucleotide sequence are greatly amplified and produce a positive, detectable detection signal, while those containing none of the target(s) nucleotide sequence are not amplified and do not produce a detection signal, or produce a signal that is below a predetermined threshold or noise level. In some embodiments, using Poisson statistics, the number of target nucleotide sequences in an original solution distributed between the reaction sites may be correlated to the number of reaction sites producing a positive detection signal. In some embodiments, the detected signal may be used to determine a number, or number range, of target molecules contained in the original solution. For example, a detection system may be configured to distinguish between reaction sites containing one target molecule and reaction sites containing two or at least two target molecules. Additionally or alternatively, the detection system may be configured to distinguish between reaction sites containing a number of target molecules that is at or below a predetermined amount and reaction sites containing more than the predetermined amount. In certain embodiments, both qPCR and dPCR processes, assays, or protocols are conducted using a single the same devices, instruments, or systems, and methods.

B. Bio-analytical Instrument

[0052] In embodiments, there are provided instruments for biological analysis with illumination systems that are capable of improving optical efficiency and reducing background signals compared to conventional bio-analytical instruments. The instrument comprises illumination system 100 or illumination system 200 as described above. In some embodiments, the instrument comprises a base configured to receive one or more sample plates containing one or more biological sample. The base may comprise a sample block assembly configured to control the temperature of one or more sample plates (e.g., sample plate 106 shown in FIG.2A and FIG. 2B). In some aspects, the base comprises a thermal cycler and the sample block comprises two or more sections with independent temperature control such that the one or more sample plates, or the zones of a sample plate can be processed with different temperatures. The base is configured to provide or perform a PCR assay.

[0053] In some instances, the sample block comprises four zones with independent temperature control. Each of the four zones of the sample block is capable of independently controlling temperature of a zone of a sample plate (e.g., zone 301, 302, 303, or 304 of sample plate 106).

[0054] According to embodiments, the base of the instrument for biological analysis can comprise a heated or temperature-controlled cover disposed over the one or more sample plates (e.g., sample plate 106). The heated or temperature-controlled cover may be used, for example, to prevent condensation above the sample contained in the sample plate, which can help maintain optical access to biological samples in the sample plate.

[0055] In some embodiments, the instrument for biological analysis comprises an optical sensor configured to receive emission from the biological samples from the one or more sample plates in response to an excitation light. Exemplary optical sensors may include a complementary metal-oxide- semiconductor sensor (CMOS), charge-coupled device (CCD) sensor, or a combination thereof. The instrument for biological analysis comprises one or more emission filters disposed between the one or more sample plates and the optical sensor. The one or more emission filters are configured, for example, to block excitation light reflected or scattered to the optical sensor. In certain embodiments, the emission filters are arranged in an emission filter wheel. In some embodiments, at least some of the emission light comprise a fluorescent emission from at least some of the biological samples in response to excitation by an excitation light.

[0056] According to embodiments, the instrument for biological analysis comprises an imaging unit configured to capture images of one or more sample plates disposed on the base. In some embodiments, the imaging unit can comprise an optical sensor circuit board and one or more sensor lenses. According to embodiments, the instrument for biological analysis can comprise one or more electronic processors configured to control, monitor, and/or receive data from the optical sensor. In some embodiments, the instrument is configured to perform a dPCR assay, and data analysis is based on images of the biological samples on the one or more sample plates.

[0057] As part of the disclosure, specific examples are included below. The examples are for illustrative purposes only and are not intended to limit the invention. Those of ordinary skill in the art will readily recognize parameters that can be changed or modified to yield essentially the same results. Example (Effects of Illumination System Configurations on Image Uniformity)

[0058] The effects of illumination system configurations, particularly the positioning of the staggered minors of illumination system 100 (as shown in FIG. 1 A), on image uniformity of a sample plate were tested.

[0059] QuantStudio™ Absolute Q^TM MAP16 plate was used as the sample plate. An LED light source was used to provide a divergent source light beam (source light beam 11). Source light beam 11 was then collimated by collimating lenses 103a and 103b shown in FIG. IB to form substantially collimated rectangular- shaped incoming light beam 13. Two configurations of staggered mirrors as shown in FIG. 2A and FIG. 2B were tested for determining the effects of the staggered mirrors configuration on image uniformity. With the staggered mirror configuration shown in FIG. 2A (configuration A), sub-beams 14a and 14d split from outer portions of incoming light beam 13 were directed to zone 303 and zone 302 respectively, both of which were disposed at the inner portions of the sample plate (sample plate 106 in FIG. 2A). The sub-beams 14b and 14c split from inner portions of incoming light beam 13 were directed to zone 304 and zone 301 respectively, both of which were disposed at the outer portions of sample plate 106 to provide oblique illumination.

[0060] With the staggered mirrors configuration shown in FIG. 2B (configuration B), subbeams 14a’ and 14d’ split from outer portions of incoming light beam 13 were directed to zone 301 and 304 respectively, both of which were disposed at the outer portions of sample plate 106. The sub-beams 14b’ and 14c’ split from inner portions of incoming light beam 13 were directed to zone 302 and zone 303 respectively, both of which were disposed at the inner portions of sample plate 106 to provide oblique illumination. Images of the sample plates loaded with FAM™ dye and VIC® dye respectively were taken with an imaging unit with a CMOS sensor.

[0061] The results are shown in FIGS. 3A-3D. FIG. 3 A shows an image taken for a FAM™ dye loaded sample plate with staggered mirrors in configuration B indicating obvious image non-uniformity across the sample plate image. FIG. 3B shows an image taken for a FAM™ dye loaded sample plate with staggered mirrors in configuration A indicating improved image uniformity via compensating the vignetting and/or roll-off effect from imaging lens. FIG. 3C shows an image taken for a VIC® dye loaded sample plate with staggered mirrors in configuration B indicating obvious non-uniformity across the sample plate image. FIG. 3D shows an image taken for a VIC® dye loaded sample plate with staggered mirrors in configuration A indicating improved image uniformity via compensating the vignetting and/or roll-off effect from imaging lens. The results indicate that by directing inner portions of a light beam to outer portions of a sample plates and outer portions of the light beam to inner portions of the sample plates via the positioning of the staggered mirrors in the disclosed illumination system, the vignetting and/or roll-off effect of imaging lens can be mitigated, thereby improving the image uniformity.

[0062] In the context of the specification, at least the following embodiments are described. Embodiment 1 is an illumination system comprising two or more staggered mirrors configured to split an incoming light beam into at least two sub-beams a guiding module configured to direct the sub-beams to provide oblique illumination to an area. Embodiment 2 is the illumination system of embodiment 1, wherein the guiding module comprises one or more front mirrors configured to reflect the sub-beams such that the sub-beams have an angle of incidence of great than 0 degrees to the area. Embodiments 3 is the illumination system of any of embodiments 1 and 2, wherein staggered mirrors are positioned such that the incoming light beam has an angle of incidence of about 45 degrees. Embodiments 4 is the illumination system of any of embodiments 1 to 3, wherein the region comprises a sample container. Embodiment 5 is the illumination system of embodiment 4, wherein the sample container includes a sample plate that contains a plurality of reaction sites comprising a biological reaction mixture. Embodiment 6 is the illumination system of embodiment 5, wherein the sample plate includes a microfluidic chip comprising a plurality of microchambers configured to contain the reaction sites. Embodiment 7 is the illumination system of any of embodiments 5 and 6, wherein the sub-beams providing the oblique illumination to the region are capable of exciting biological samples of the reaction sites to produce emission light. Embodiment 8 is the illumination system of embodiment 7, further comprising an optical sensor configured to receive the emission light from the sample plate. Embodiment 9 is the illumination system of any of embodiments 5 to 8, wherein the sample plate is disposed below a manifold, wherein the manifold is configured to secure positioning of the sample plate. Embodiment 10 is the illumination system of any of embodiment 9, wherein the manifold is configured to provide gas exchange mechanism for the sample plate. Embodiment 11 is illumination system of any of embodiments 5 to 10, wherein the sample plate includes at least two zones, each of two adjacent zones are separated by a divider. Embodiment 12 is the illumination system of embodiment 11, wherein each of the at least two zones of the sample plate receives at least one sub-beam from the staggered mirrors. Embodiment 13 is the illumination system of embodiments 11 and 12, wherein the dividing area receives substantially no illumination directly from the sub-beams. Embodiment 14 is the illumination system of any of embodiments 6 to 13, wherein the illumination efficiency of the illumination system is from 15 to 30%. Embodiment 15 is the illumination system of any of embodiments 1 to 14, further comprising a collimator configured to collimate a narrowed light beam to produce the incoming light beam. Embodiment 16 is the illumination system of embodiment 15, wherein the collimator comprises at least two cylindrical collimating lenses with different focal lengths, configured to generate the incoming beam. Embodiment 17 is the illumination system of embodiment 15, wherein the collimator comprises a single collimating lens. Embodiment 18 is the illumination system of any of embodiments 15 to 17, further comprising a beam shaper configured to shape a source light beam to produce the narrowed light beam. Embodiment 19 is the illumination system of embodiment 18, wherein the source light beam comprises a divergent LED beam, a laser light beam, a xenon light beam, a laser beam, an argon light beam, a krypton light beam, a tungsten halogen light beam, an incandescent light beam, or a light beam from any other type of commercially available light source. Embodiment 20 is the illumination system of embodiment 19, wherein the source light beam is produced by an LED source, and a distance from the LED source to the obliquely illuminated region is no more than 1200 mm. Embodiment 21 is the illumination system of any of embodiments 20, wherein the light path from the LED source to the obliquely illuminated region is from 600 to 1200 mm. Embodiment 22 is the illumination system of any of embodiments 1 to 21, wherein the staggered mirrors are configured to split the incoming beam with substantially no beam loss. Embodiment 23 is the illumination system of any of embodiments 1 to 22, wherein the illumination system is configured to provide illumination and/or excitation light for a digital Polymerase Chain Reaction (dPCR) assay.

[0063] Embodiment 24 is an illumination system comprising n staggered mirrors configured to split an incoming light beam into m sub-beams; a guiding module configured to direct the sub-beams to provide oblique illumination to a sample plate, wherein the sample plate comprises k zones, each zone comprising a plurality of reaction sites; and wherein n, m, and k, are each an integer, and m > k, 2<m<n. Embodiment 25 is the illumination system of embodiment 24, wherein the reaction sites comprise a reaction mixture that contains a biological sample. Embodiment 26 is the illumination system of embodiment 25, wherein the sub-beams are capable of exciting the biological sample to generate emission light. Embodiment 27 is the illumination system of embodiment 26, wherein the sub-beams providing oblique illumination to the sample plate are not parallel to the emission light. Embodiment 28 is the illumination system of embodiment 27, wherein the sub-beams providing oblique illumination to the sample plate have an angle of incidence of 25 to 40 degrees. Embodiment 29 is the illumination system of embodiment 27, wherein the sub-beams providing oblique illumination to the sample plate have an angle of incidence of about 32 degrees. Embodiment 30 is the illumination system of embodiments 24 to 29, wherein the sample plate comprises a microfluidic chip. Embodiment 31 is the illumination system of embodiment 30, wherein the microfluidic chip comprises a plurality of microchambers. Embodiment 32 is he illumination system of embodiment 31, wherein at least some of the microchambers each contain a reaction site. Embodiment 33 is the illumination system of any of embodiments 31 and 32, wherein the sample plate comprises at least 20,000 reaction sites. Embodiment 34 is the illumination system of any of embodiments 24 to 33, wherein the sample plate comprises a plurality of zones, each zone comprises a plurality of reaction sites. Embodiment 35 is the illumination system of embodiment 34, wherein adjacent two zones are separated by a divider, and the divider receives substantially no illumination from the sub-beams. Embodiment 36 is the illumination system of embodiment 35, wherein the staggered mirrors are configured such that each sub-beam provides illumination to one zone. Embodiment 37 is the illumination system of embodiment 36, wherein the staggered mirrors are configured such that mirrors receiving inner portions of the incoming light beam direct corresponding sub-beams to the zones disposed at outer portions of the sample plates. Embodiment 38 is the illumination system of embodiment 37, wherein the staggered mirrors are arranged to compensate for vignetting and/or roll-off effect of an imaging lens when an image is taken for the sample plate, thereby improving image uniformity of the sample plate. Embodiment 39 is the illumination system of any of embodiments 35 to 38, wherein the staggered mirrors are not in the same plane, and a distance between the staggered mirrors are determined by positioning of the zones of the sample plate. Embodiment 40 is the illumination system of any of embodiments 24 to 39, further comprising a beam shaper configured to shape a source light beam to produce a narrowed light beam; and one or more collimators configured to collimate the narrowed light beam to produce the incoming light beam. Embodiment 41 is the illumination system of embodiment 40, wherein the beam shaper is a beam shaper lens. Embodiment 42 is the illumination system of any of embodiments 40 and 41, wherein the collimator comprises a collimating lens. Embodiment 43 is the illumination system of any of embodiments 40 and 41, wherein the collimator comprises at least two cylindrical collimating lenses with different focal lengths, configured to generate a rectangular incoming light beam. Embodiment 44 is the illumination system of any of embodiments 40 to 43, wherein the oblique illumination of the sample plate is capable of improving optical efficiency of the illumination system compared to on-axis illumination. Embodiment 45 is the illumination system of any of embodiments 40 to 44, further comprising a light source configured to provide the source light beam. Embodiment 46 is the illumination system of embodiment 45, wherein the light source comprises an LED light source, a laser light source, a halogen light source, a tungsten light source, a xenon light source, an argon light source, a krypton light source, an incandescent light source, or a combination thereof. Embodiment 47 is the illumination system of any of embodiments 45 and 46, wherein light path from the light source to the sample plate is from about 600 to 1200 mm. Embodiment 48 is the illumination system of any of embodiments 24 to 47, wherein optical efficiency for the illumination system is from about 15% to 35%. Embodiment 49 is the illumination system of any of embodiments 24 to 48, wherein the guiding module comprises one or more front mirrors configured to reflect the sub-beams to the sample plate at an angle of incidence of 25 to 40 degrees. Embodiment 50 is the illumination system of embodiment 49, wherein the guiding module comprises a single front mirror. Embodiment 51 is the illumination system of embodiment 49, wherein the guiding module comprises n front mirrors, each front mirror corresponding to a staggered mirror. Embodiment 52 is the illumination system of any of embodiments 24 to 51, wherein the illumination system is an illumination system for a digital PCR system.

[0064] Embodiment 53 is an instrument for biological analysis, the instrument comprising a base configured to receive one or more sample plates containing a biological sample; an illumination system configured to illuminate the one or more sample plates. The illumination system comprises n staggered mirrors configured to split an incoming light beam into m subbeams: a front mirror configured to direct the sub-beams to provide oblique illumination to the one or more sample plates and/or to excite the biological samples to produce emission light, wherein the incoming light beam and the sub-beams are collimated light beams; wherein the sample plate comprises k zones, each zone comprises a plurality of reaction sites, where n, m, and k, are each an integer, and n > k, 2<m<n. The instrument comprises an optical sensor configured to receive the emission light from the biological samples. Embodiment 54 is the instrument of embodiment 53, wherein the base further comprises a thermal cycler configured to perform a polymerase chain reaction on the biological samples. Embodiment 55 is the instrument of any of embodiments 53 and 54, wherein the instrument is configured to conduct a digital PCR assay. Embodiment 56 is the instrument of any of embodiments 53 to 55, wherein the sample plate is a microfluidic chip comprising at least 20,000 partitions. Embodiment 57 is the instrument of embodiment 56, wherein the microfluidic chip comprises at least 20,000 microchambers. Embodiment 58 is the instrument of any of embodiments 53 to 57, further comprising one or more emission filters disposed between the one or more sample plates and the optical sensor. Embodiment 59 is the instrument of embodiment 58, wherein at least some of the emission light comprise a fluorescent emission from at least some of the biological samples in response to excitation by an excitation light. Embodiment 60 is the instrument of embodiment 59, further comprising an imaging unit configured to capture images of the sample plates. Embodiment 61 is the instrument of any of embodiments 48 to 55, wherein the illumination system further comprises a light source configured to provide source light beam; a beam shaper configured to shape a source light beam to produce a narrowed light beam; and one or more collimators configured to collimate the narrowed light beam to produce the incoming light beam. Embodiment 62 is the instrument of embodiment 56, wherein a light path from the light source to the one or more sample plates is from about 600 to 1200 mm. Embodiment 63 is the instrument of any of embodiments 48 to 57, wherein the optical sensor comprises a complementary metal-oxide-semiconductor sensor.

[0065] Although embodiments of the present application and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the embodiments as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the system, instrument, machine, manufacture, composition of matter, means, methods, and/or steps described in the specification. As one of ordinary skill in the art will readily appreciate from the above disclosure, system, instrument, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims

CLAIMS What is claimed is:

1. An illumination system comprising: two or more staggered mirrors configured to split an incoming light beam into at least two sub-beams; and a guiding module configured to direct the sub -beams to provide oblique illumination to an area.

2. The illumination system of claim 1, wherein the guiding module comprises one or more front mirrors configured to reflect the sub-beams such that the sub-beams have an angle of incidence of great than 0 degrees to the area.

3. The illumination system of any of claims 1 and 2, wherein staggered mirrors are positioned such that the incoming light beam has an angle of incidence of about 45 degrees.

4. The illumination system of any of claims 1 to 3, wherein the area comprises a sample holder.

5. The illumination system of claim 4, wherein the sample holder includes a sample plate that contains a plurality of reaction sites comprising a biological reaction mixture.

6. The illumination system of claim 5, wherein the sample plate includes a microfluidic chip comprising a plurality of microchambers configured to contain the reaction sites.

7. The illumination system of any of claims 5 and 6, wherein the sub-beams providing the oblique illumination to the sample plate are capable of exciting biological samples of the reaction sites to produce emission light.

8. The illumination system of claim 7, further comprising an optical sensor configured to receive the emission light from the sample plate.

9. The illumination system of any of claims 5 to 8, wherein the sample plate is disposed below a manifold, wherein the manifold is configured to secure positioning of the sample plate.

10. The illumination system of claim 9, wherein the manifold is configured to provide gas exchange mechanism for the sample plate.

11. The illumination system of any of claims 5 to 10, wherein the sample plate includes at least two zones, each of two adjacent zones are separated by a dividing area.

12. The illumination system of claim 11, wherein each of the at least two zones of the sample plate is configured to receive at least one sub-beam from the staggered mirrors.

13. The illumination system of any of claims 11 and 12, wherein the dividing area receives substantially no illumination directly from the sub-beams.

14. The illumination system of any of claims 6 to 13, wherein the illumination efficiency of the illumination system is from 15 to 30%.

15. The illumination system of any of claims 1 to 14, further comprising: a collimator configured to collimate a narrowed light beam to produce the incoming light beam.

16. The illumination system of claim 15, wherein the collimator comprises at least two cylindrical collimating lenses with different focal lengths, configured to generate the incoming beam.

17. The illumination system of claim 15, wherein the collimator comprises a single collimating lens.

18. The illumination system of any of claims 15 to 17, further comprising: a beam shaper configured to shape a source light beam to produce the narrowed light beam.

19. The illumination system of claim 18, wherein the source light beam comprises a divergent LED beam, a laser light beam, a xenon light beam, a laser beam, an argon light beam, a krypton light beam, a tungsten halogen light beam, an incandescent light beam, or a combination thereof.

20. The illumination system of claim 19, wherein the source light beam is produced by an LED source, and a distance from the LED source to the obliquely illuminated area is no more than 1200 mm.

21. The illumination system of any of claims 20, wherein the light path from the LED source to the obliquely illuminated area is from 600 to 1200 mm.

22. The illumination system of any of claims 1 to 21, wherein the staggered mirrors are configured to split the incoming beam with substantially no beam loss.

23. The illumination system of any of claims 1 to 22, wherein the illumination system is configured to provide illumination and/or excitation light for a digital Polymerase Chain Reaction (dPCR) assay.

24. An illumination system comprising: n staggered mirrors configured to split an incoming light beam into m sub-beams; a guiding module configured to direct the sub -beams to provide oblique illumination to a sample plate; wherein the sample plate comprises k zones, each zone comprising a plurality of reaction sites; and wherein n, m, and k, are each an integer, and m > k. 2<m<n.

25. The illumination system of claim 24, wherein the reaction sites comprise a reaction mixture that contains a biological sample.

26. The illumination system of claim 25, wherein the sub-beams are capable of exciting the biological sample to generate emission light.

27. The illumination system of claim 26, wherein the sub-beams providing oblique illumination to the sample plate are not parallel to the emission light.

28. The illumination system of claim 27, wherein the sub-beams providing oblique illumination to the sample plate have an angle of incidence of 25 to 40 degrees.

29. The illumination system of claim 27, wherein the sub-beams providing oblique illumination to the sample plate have an angle of incidence of about 32 degrees.

30. The illumination system of claims 24 to 29, wherein the sample plate comprises a microfluidic chip.

31. The illumination system of claim 30, wherein the microfluidic chip comprises a plurality of microchambers.

32. The illumination system of claim 31, wherein each of at least some of the microchambers contains a reaction site.

33. The illumination system of any of claims 31 and 32, wherein the sample plate comprises at least 20,000 reaction sites.

34. The illumination system of any of claims 24 to 33, wherein the sample plate comprises a plurality of zones, each zone comprises a plurality of reaction sites.

35. The illumination system of claim 34, wherein two adjacent zones are separated by a divider, and the divider receives substantially no illumination from the sub-beams.

36. The illumination system of claim 35, wherein the staggered mirrors are configured such that each sub-beam provides illumination to one zone.

37. The illumination system of claim 36, wherein the staggered mirrors are configured such that mirrors receiving inner portions of the incoming light beam direct corresponding sub-beams to the zones disposed at outer portions of the sample plates.

38. The illumination system of claim 37, wherein the staggered mirrors are arranged to compensate for vignetting and/or roll-off effect of an imaging lens when an image is taken for the sample plate, thereby improving image uniformity of the sample plate.

39. The illumination system of any of claims 35 to 38, wherein the staggered mirrors are not in the same plane, and a distance between the staggered mirrors are determined by positioning of the zones of the sample plate.

40. The illumination system of any of claims 24 to 39, further comprising: a beam shaper configured to shape a source light beam to produce a narrowed light beam; and one or more collimators configured to collimate the narrowed light beam to produce the incoming light beam.

41. The illumination system of claim 40, wherein the beam shaper is a beam shaper lens.

42. The illumination system of any of claims 40 and 41, wherein the collimator comprises a collimating lens.

43. The illumination system of any of claims 40 and 41, wherein the collimator comprises at least two cylindrical collimating lenses with different focal lengths, configured to generate a rectangular incoming light beam.

44. The illumination system of any of claims 40 to 43, wherein the oblique illumination of the sample plate is configured to improve optical efficiency of the illumination system compared to on-axis illumination.

45. The illumination system of any of claims 40 to 44, further comprising a light source configured to provide the source light beam.

46. The illumination system of claim 45, wherein the light source comprises an LED light source, a laser light source, a halogen light source, a tungsten light source, a xenon light source, an argon light source, a krypton light source, an incandescent light source, or a combination thereof.

47. The illumination system of any of claims 45 and 46, wherein light path from the light source to the sample plate is from about 600 to 1200 mm.

48. The illumination system of any of claims 24 to 47, wherein optical efficiency for the illumination system is from about 15% to 35%.

49. The illumination system of any of claims 24 to 48, wherein the guiding module comprises one or more front mirrors configured to reflect the sub-beams to the sample plate at an angle of incidence of 25 to 40 degrees.

50. The illumination system of claim 49, wherein the guiding module comprises a single front mirror.

51. The illumination system of claim 49, wherein the guiding module comprises n front mirrors, and each front mirror corresponds to a staggered mirror.

52. The illumination system of any of claims 24 to 51, wherein the illumination system is an illumination system for a digital PCR system.

53. An instrument for biological analysis, the instrument comprising: a base configured to receive one or more sample plates containing a biological sample; an illumination system configured to illuminate the one or more sample plates, wherein the illumination system comprises: n staggered mirrors configured to split an incoming light beam into m sub-beams; a front mirror configured to direct the sub-beams to provide oblique illumination to the one or more sample plates and/or to excite the biological samples to produce emission light beams, wherein the incoming light beam and the sub-beams are substantially collimated light beams; wherein the sample plate comprises k zones, each zone comprises a plurality of reaction sites, where n, m, and k, are each an integer, and m > k, 2< <n; and an optical sensor configured to receive the emission light from the biological samples.

54. The instrument of claim 53, wherein the base further comprises a thermal cycler configured to perform a polymerase chain reaction on the biological samples.

55. The instrument of any of claims 53 and 54, wherein the instrument is configured to conduct a digital PCR assay.

56. The instrument of any of claims 53 to 55, wherein the sample plate comprises a microfluidic chip configured to segregate a sample into at least 20,000 partitions.

57. The instrument of claim 56, wherein the microfluidic chip comprises at least 20,000 microchambers.

58. The instrument of any of claims 53 to 57, further comprising one or more emission filters disposed between the one or more sample plates and the optical sensor.

59. The instrument of claim 58, wherein at least some of the emission light comprises a fluorescent emission from at least some of the biological samples in response to excitation by an excitation light.

60. The instrument of claim 59, further comprising an imaging unit configured to capture images of the sample plates.

61. The instrument of any of claims 48 to 55, wherein the illumination system further comprises: a light source configured to provide source light beam; a beam shaper configured to shape a source light beam to produce a narrowed light beam; and one or more collimators configured to collimate the narrowed light beam to produce the incoming light beam.

62. The instrument of claim 56, wherein a light path from the light source to the one or more sample plates is from about 600 to 1200 mm.

63. The instrument of any of claims 48 to 57, wherein the optical sensor comprises g complementary meta! -oxide-semiconductor sensor (CMOS), or a charge-coupled device (CCD) sensor.