Illumination system for a biological analysis instrument

Abstract

A lighting system and a bioanalytical instrument comprising the same are disclosed. The lighting system comprises two or more interleaved mirrors configured to split an incoming light beam into at least two sub-beams, and a guiding module configured to guide the sub-beams to provide oblique illumination to an area. Compared to conventional lighting systems for bioanalytical instruments, the lighting system is capable of improving illumination efficiency, reducing background signal and improving image uniformity.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 603,345, filed November 28, 2023, which is incorporated herein by reference. Technical Field

[0003] This disclosure relates to optical systems. More specifically, this disclosure relates to an illumination system for a bioanalytical instrument. Even more specifically, this disclosure relates to an illumination system comprising staggered mirrors for splitting source light and providing oblique illumination to a biological sample. Background Technology

[0004] Conventional illumination systems for bioanalytical instruments are typically configured to provide coaxial illumination of the biological sample, where the excitation beam and / or illumination beam are coaxial with the emission from the sample. In these systems, a dichroic beam splitter or a 50 / 50 beam splitter is used to reflect the illumination and / or excitation beam onto the sample, directing the emitted light toward the optical detector or camera. However, coaxial illumination often results in more light being reflected and scattered from the sample holder into the optical detector or camera, leading to a higher background signal during data acquisition and / or image capture of the biological sample.

[0005] Furthermore, when an optical system uses a single 50 / 50 beamsplitter, at least 75% of optical power (watts) is lost. This can severely limit data accuracy when used to illuminate samples with low sample volumes, low concentrations, and / or low emission intensities. On the other hand, while dichroic beamsplitters offer high optical efficiency, each optical channel is coupled to a dichroic beamsplitter, significantly increasing the footprint and overall cost of bioanalytical instruments that typically have multiple optical channels. Moreover, existing optical systems for bioanalytical instruments indiscriminately illuminate both sample-containing and sample-free areas of the sample holder / plate, potentially leading to wasted optical power (watts) and high background signals reflected and scattered by sample-free areas.

[0006] Overall, while lighting systems exist for bioanalytical instruments, the need for improvement in this field remains due to at least the aforementioned shortcomings of these systems. Summary of the Invention

[0007] Solutions have been found to at least some of the aforementioned problems related to the illumination systems of bioanalytical instruments. The solution lies in an illumination system comprising staggered mirrors configured to split a source beam into multiple sub-beams, which are then directed to sample-containing areas of the sample holder. This helps to avoid illuminating sample-free areas of the sample holder, thereby reducing energy consumption and light source footprint.

[0008] Furthermore, by avoiding illumination of non-sample areas of the sample holder, the staggered reflectors can significantly improve illumination efficiency, allowing the illumination system disclosed herein to be used for illuminating small sample volumes, low sample concentrations, and / or low emission intensities. For example, the illumination system disclosed herein can be used for digital PCR assays that require high illumination efficiency because each reaction site and / or sample partition of the sample plate contains low concentrations and / or low volumes of sample / probe.

[0009] In addition, the illumination system includes a guidance module for directing sub-beams from staggered mirrors toward the biological sample for tilted illumination. Notably, compared to existing coaxial illumination systems, implementing tilted illumination reduces background signal, resulting in higher image and / or data quality when acquiring sample images.

[0010] Furthermore, the staggered reflectors of the illumination system disclosed herein can be configured to guide the inner portion of the source beam to illuminate the outer portion of the sample plate, such that the outer portion of the sample plate receives a higher light intensity than the inner portion. This can mitigate vignetting and / or attenuation effects in the image of the sample plate, resulting in higher image uniformity and improved image-based data quality. Therefore, the illumination system disclosed herein provides a technical solution to at least some of the problems associated with the aforementioned currently available illumination systems.

[0011] Some embodiments relate to an illumination system. In some aspects, the illumination system includes two or more staggered reflectors configured to split an incident light beam into at least two sub-beams. The illumination system includes a guiding module configured to guide the sub-beams to provide oblique illumination to a region. This region may include a sample plate for a biological sample.

[0012] Some embodiments relate to lighting systems. In some aspects, the lighting system includes... n A series of staggered reflectors, configured to split the incident light beam into... m Sub-beams. The illumination system includes a guide module configured to guide the sub-beams to provide oblique illumination to the sample plate. The sample plate includes... k Each region contains multiple reaction sites. n, m and k Each is an integer, and m ≥ k ,2≤ m ≤ n .

[0013] Some embodiments relate to instruments for bioanalysis. In some aspects, the instrument includes a base configured to receive one or more sample plates containing biological samples. The instrument includes an illumination system configured to illuminate the one or more sample plates. The illumination system includes... n A series of staggered reflectors, configured to split the incident light beam into... m Sub-beams. The illumination system includes one or more front mirrors configured to guide the sub-beams to provide oblique illumination to one or more sample plates and / or excite biological samples to generate emitted light. In some cases, the incident beam and sub-beams are substantially collimated or collimated beams. The sample plates include... k Each region contains multiple reaction sites, among which... n, m and k Each is an integer, and m ≥ k ,2≤ m ≤ n The instrument includes an optical sensor configured to receive emitted light from a biological sample.

[0014] The following includes definitions of various terms and phrases used throughout this specification.

[0015] The definition of the terms "about" or "approximately" is close to the understanding of those skilled in the art. In a non-limiting embodiment, the term is defined as less than 10%, preferably less than 5%, more preferably less than 1%, and most preferably less than 0.5%.

[0016] The term “substantially” and its variants are defined as the range that includes within 10%, within 5%, within 1%, or within 0.5%.

[0017] As used herein, “emission” or “emitted light” means electromagnetic radiation generated by the interaction of radiation from an excitation source (e.g., excitation light) with one or more samples containing or believed to contain one or more chemical and / or biological molecules or compounds of interest. Emission may be attributed to reflection, refraction, polarization, absorption, and / or other optical effects of the sample on the radiation from the excitation source. For example, emission may include luminescence or fluorescence caused by the absorption of excitation electromagnetic radiation (e.g., excitation light) by one or more samples.

[0018] As used herein, a lens refers to an optical element configured to guide or focus incident electromagnetic radiation in order to converge or diverge such radiation, for example, to provide a real or virtual image at a finite distance or optical infinity. A lens may comprise a single optical element having focusing capabilities provided by the refraction, reflection, and / or diffraction of the incident electromagnetic radiation. Alternatively, the lens may comprise a composite system containing multiple optical elements, such as, but not limited to, achromatic lenses, doublet lenses, triplet lenses, or camera lenses. The lens may be at least partially housed in a lens case or lens mount, or at least partially encapsulated by a lens case or lens mount.

[0019] As used herein, the term "biological sample" means a sample or solution containing any type of biochemical substance or component and / or any target molecule of interest to a user, manufacturer, or distributor of the various embodiments of the invention described or implied herein, as well as any sample or solution containing an associated chemical substance or compound for performing a bioassay, experiment, or test. These biochemical substances, 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 include one or more of the following: 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 quantitative standards, genotyping, sequencing assays, experiments or protocols, sequencing validation, mutation detection, detection of genetically modified organisms, detection of rare alleles, and / or copy number changes.

[0020] The terms “beam,” “light beam,” and their variations are defined as the directional projection of light energy radiated from a light source.

[0021] The term "interlaced mirrors" and its variations are defined as mirrors that are offset from each other in at least one direction.

[0022] The term "collimation" and its variations, as disclosed throughout the specification, are defined as making the rays of a beam or particles substantially parallel at a maximum angle of 3 or 5 degrees or greater, depending on specific requirements, including illumination uniformity or image uniformity requirements. In some respects, one or more collimating lenses may be used to collimate the beam.

[0023] The terms “oblique illumination,” “off-axis illumination,” and variations thereof, disclosed throughout the specification, are defined as illumination light that is directed at an angle rather than perpendicular to the sample or specimen.

[0024] The term “illuminance efficiency” and its variations, as disclosed throughout the specification, are defined as the light power (watts) received by the sample-containing area of ​​the sample holder or sample plate and the total light power (watts) emitted by the light source of the sample holder or sample plate in the illumination system.

[0025] The terms “suppress” or “reduce” or “prevent” or “avoid” or any variations thereof, when used in the claims and / or specification, include any measurable reduction or complete suppression to achieve the desired result.

[0026] The term “effective” as used in this specification and / or claims means sufficient to achieve the desired, expected, or anticipated result.

[0027] When used in the claims or description with the terms “comprising,” “including,” “containing,” or “having,” the use of the word “a” or “an” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more.”

[0028] The words “comprising” (and any form of inclusion, 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, unlisted elements or steps of the process.

[0029] The systems and / or instruments disclosed herein may “comprise”, “compose of” or “are composed of” specific components, compositions, etc., disclosed throughout the specification. Regarding the transitional phase “composes of”, in a non-limiting aspect, a fundamental and novel feature of the lighting systems disclosed herein is staggered reflectors configured to split an incident beam into sub-beams and guide the sub-beams to provide oblique illumination to an area.

[0030] Other objects, features, and advantages of the embodiments will become apparent from the following accompanying drawings, detailed description, and examples. However, it should be understood that while the drawings, detailed description, and examples indicate specific embodiments, they are given by way of illustration only and are not intended to be limiting. Furthermore, changes and modifications within the spirit and scope of this detailed description will become apparent to those skilled in the art. In other embodiments, features from a particular embodiment may be combined with features from other embodiments. For example, a feature from one embodiment may be combined with features from any of the other embodiments. In other embodiments, additional features may be added to the specific embodiments described herein. Attached Figure Description

[0031] For a more complete understanding, please refer to the following description in conjunction with the accompanying drawings, in which:

[0032] Figure 1A An illumination system comprising two collimating lenses is shown according to an embodiment disclosed in the specification;

[0033] Figure 1B An illumination system including a single collimating lens is shown according to an embodiment disclosed in the specification;

[0034] Figure 2A and Figure 2B Two different configurations of staggered reflectors according to embodiments disclosed in this specification are shown, which guide different portions of an incident beam to provide illumination to the inner and outer portions of a sample plate; Figure 2A The diagram shows a configuration of staggered reflectors used to guide the outer portion of an incident beam to the inner portion of a sample plate and vice versa. Figure 2B The diagram shows a configuration of staggered reflectors used to guide the outer portion of the incident beam to the outer portion of the sample plate and the inner portion of the incident beam to the outer portion of the sample plate.

[0035] Figures 3A to 3D Images of a sample plate captured using the lighting system disclosed in this specification are shown. Figure 3A Showing the use of Figure 2B The staggered mirror configuration shown is for FAM TM Images acquired from dye-loaded sample plates; Figure 3B Showing the use of Figure 2A The staggered mirror configuration for FAM TM Images acquired from dye-loaded sample plates; Figure 3C Showing the use of Figure 2B The staggered configuration of the mirrors for VIC ®Images acquired from dye-loaded sample plates; Figure 3D Showing the use of Figure 2A Images were acquired from a VIC dye-loaded sample plate using an array of staggered mirrors. Detailed Implementation

[0036] Current illumination systems for bioanalytical instruments suffer from several drawbacks, including low illumination efficiency, high background signal, large footprint, and / or high manufacturing cost. Embodiments of the present invention provide solutions to at least some of these problems. These solutions presuppose an illumination system comprising two or more staggered mirrors configured to split an incident beam into multiple sub-beams and guide these sub-beams to the sample-containing area of ​​the sample holder, thereby improving illumination efficiency and reducing the required power and light source size of the illumination system. Furthermore, the illumination system disclosed herein includes a guiding module configured to guide the sub-beams from the staggered mirrors to provide tilted illumination to the sample plate, resulting in reduced background signal. Additionally, the staggered mirrors of the illumination system can be configured to mitigate vignetting and / or attenuation effects in the sample image, which can improve image uniformity and data quality in bioanalytical instruments.

[0037] These and other non-limiting aspects of the invention are discussed in more detail in the following sections.

[0038] A. Lighting system

[0039] Illumination systems in bioanalytical instruments are typically designed to illuminate biological samples for imaging and / or excitation. For conventional illumination systems in bioanalytical instruments, particularly polymerase chain reaction (PCR) instruments, coaxial illumination is used because the sample / probe concentration, sample volume, and inherent emission intensity of conventional PCR or qPCR are sufficient to overcome the low illumination efficiency and high background signal associated with coaxial illumination. However, when sample concentration, sample volume, and / or emission intensity are relatively low, such as in digital PCR assays, currently available illumination systems can be disadvantageous in terms of image and / or data quality. The illumination system disclosed in this specification provides tilted (off-axis) illumination of the sample, thereby improving image uniformity and / or data quality in bioassays.

[0040] refer to Figure 1AThe diagram illustrates a schematic of an illumination system 100, which can be used to provide illumination and / or excitation for biological samples. According to an embodiment, the illumination system 100 includes a light source 101 configured to provide a source beam 11. The light source 101 can be of any shape, including circular, square, rectangular, triangular, fan-shaped, irregular shapes, or any combination thereof. In some embodiments, the light source 101 includes a light-emitting diode (LED) light source, an organic LED (OLED) light source, a tungsten halide 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. The light source 101 can have a power of 6 watts to 8 watts and all ranges or values ​​therebetween at a given drive current, including ranges of 6 watts to 6.2 watts, 6.2 watts to 6.4 watts, 6.4 watts to 6.6 watts, 6.6 watts to 6.8 watts, 6.8 watts to 7.0 watts, 7.0 watts to 7.2 watts, 7.2 watts to 7.4 watts, 7.4 watts to 7.6 watts, 7.6 watts to 7.8 watts, and 7.8 watts to 8.0 watts. The light source 101 can have a wide spectral range required by bioanalytical instruments. The light source 101 can have a balanced optical power (watt) distribution across the spectrum. In some embodiments, the source beam 11 is a diverging beam.

[0041] In some embodiments, the illumination system 100 includes a beam shaper 102 configured to narrow a source beam 11 and / or shape the source beam to produce a narrowed beam 12. The illumination system 100 may include a collimator 103 configured to collimate the narrowed beam 12 to produce an incident beam 13. The incident beam 13 is a collimated or substantially collimated beam. In some embodiments, the collimator 103 of the illumination system 100 includes two cylindrical collimating lenses (103a and 103b) with different focal lengths, configured to generate a rectangular incident beam 13. In some embodiments, both cylindrical collimating lenses 103a and 103b are rectangular. In some respects, the collimating lens 103a has a focal length of 350 mm to 450 mm and all ranges and values ​​therebetween, including the 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 and 440 mm to 450 mm. Collimating lens 103b has a focal length of 450 mm to 550 mm and all ranges and values ​​therebetween, including the ranges of 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. Rectangular collimating lenses 103a and 103b are positioned in different orientations such that the incident beam is shaped into a rectangular shape. For example, in some embodiments, rectangular collimating lens 103a is positioned such that its convex edge is in the vertical direction, and rectangular collimating lens 103b is positioned such that its convex edge is in the horizontal direction. Collimating lenses 103a and 103b are made as a combination of a concave lens of borosilicate glass (e.g., Schott BK7®), plastic, flint glass, and a convex lens of coronal glass as an achromatic doublet, or a combination thereof. In some respects, collimating lenses 103a and 103b have a wavelength range of 400 nm to 800 nm.According to the embodiment, the optical path from the light source 101 to the collimator 103 is in the range of 350 mm to 550 mm, and all ranges and values ​​therebetween, including 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.

[0042] In some embodiments, the lighting system 100 includes staggered reflectors 104 (e.g., such as...). Figure 1A As shown in 104a, 104b, 104c, and 104d, these mirrors are configured to split the incident beam 13 into a plurality of sub-beams 14 (e.g., 14a, 14b, 14c, and 14d). Beam splitting by the staggered mirrors 104 involves each of the staggered mirrors reflecting a portion of the incident beam 13, such that each reflected portion of the incident beam 13 forms a sub-beam (e.g., 14a, 14b, 14c, or 14d). According to the embodiments disclosed in this specification, the staggered mirrors 104 are configured such that there is substantially no light loss when the incident beam 13 is split into sub-beams 14. The sub-beams 14 may be substantially parallel to each other. Each sub-beam of the sub-beams 14 is a collimated or substantially collimated beam.

[0043] The staggered mirrors 104 include optical mirrors. The optical mirrors have a reflectivity that reflects light. The staggered mirrors 104 (e.g., 104a, 104b, 104c, and 104d) are not in the same plane. The staggered mirrors 104 (e.g., 104a, 104b, 104c, and 104d) may be substantially parallel to each other. In some respects, the staggered reflectors 104 are positioned such that the incident beam 13 has an angle of incidence of 30 to 60 degrees relative to the staggered reflectors 104, and all values ​​and ranges therebetween, including 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 cases, the angle of incidence of the incident beam 13 relative to the staggered reflectors 104 is 45 degrees.

[0044] According to embodiments disclosed in this specification, the lighting system 100 includes a guiding module 105 configured to guide sub-beams 14 (e.g., 14a-14d) to provide oblique illumination to an area. In some cases, the guiding module 105 includes one or more front reflectors configured to reflect the sub-beams 14 (e.g., 14a-14d) to generate reflected sub-beams 15 (e.g., 15a-15d), such that the reflected sub-beams 15 (e.g., 15a-15d) provide oblique illumination to the area. According to some embodiments, the reflected sub-beams 15 (e.g., 15a-15d) are collimated or substantially collimated beams. In some aspects, the sub-beams 14 (e.g., 14a-14d) have an angle of incidence relative to the front reflector ranging from 20 degrees to 40 degrees, and all ranges and values ​​therebetween, including 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 cases, the guide module 105 includes a single front reflector, and the sub-beams 14 have an angle of incidence of 32 degrees relative to the front reflector. In some other cases, the guide module 105 includes multiple front reflectors, and the sub-beams 14 (e.g., 14a-14d) have an angle of incidence of 20-40 degrees relative to the front reflectors. The incident angle of the reflector beam 15 relative to the region is 20 degrees to 40 degrees, and all ranges and values ​​therebetween, including the ranges of 20 degrees to 22 degrees, 22 degrees to 24 degrees, 24 degrees to 26 degrees, 26 degrees to 28 degrees, 28 degrees to 30 degrees, 30 degrees to 32 degrees, 32 degrees to 34 degrees, 34 degrees to 36 degrees, 36 degrees to 38 degrees, and 38 degrees to 40 degrees. In some embodiments, the optical path from the staggered reflectors 104 to one or more front reflectors of the guide module 105 is 200 mm to 400 mm and all ranges and values ​​therebetween, including the ranges of 200 mm to 220 mm, 220 mm to 240 mm, 240 mm to 260 mm, 260 mm to 280 mm, 280 mm to 300 mm, 300 mm to 320 mm, 320 mm to 340 mm, 340 mm to 360 mm, 360 mm to 380 mm, and 380 mm to 400 mm.

[0045] In some embodiments, the area illuminated by the reflector beams 15 (e.g., 15a-15d) includes the sample plate 106. The sample plate 106 may include multiple zones. Each zone of the sample plate 106 includes multiple reaction sites. In embodiments, any two adjacent zones of the sample plate 106 are separated by separators. Each separator receives significantly less illumination directly from the reflector beams 15 (e.g., reflector beams 15a-15d). In some cases, each separator 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 the reflector beams (e.g., reflector beams 15a-15d) compared to each zone of the sample plate. In some embodiments, each separator receives substantially no illumination directly from any of the reflector beams 15 (e.g., reflector beams 15a-15d). According to embodiments, the optical path from the light source 101 to the sample plate 106 is 600 mm to 1200 mm, and all values ​​and ranges therebetween, including the ranges of 600 mm to 650 mm, 650 mm to 700 mm, 700 mm to 750 mm, 750 mm to 800 mm, 800 mm to 850 mm, 850 mm to 900 mm, 900 mm to 950 mm, 950 mm to 1000 mm, 1000 mm to 1050 mm, 1050 mm to 1100 mm, 1100 mm to 1150 mm, and 1150 mm to 1200 mm. In some cases, the optical path from the light source 101 to the sample plate 106 is approximately 900 mm.

[0046] In some embodiments, the lighting system 100 includes n An interlaced reflector, and the incident beam 13 is split into... m Individual beam 14, and sample plate 106 including k Each district, among which n, m and k Each is an integer, and m ≥ k ,2≤ m ≤ n In some aspects, one or more of the sub-beams are configured to provide oblique illumination to a single region of the sample plate 106. Each region of the sample plate 106 includes multiple reaction sites. In some cases, staggered mirrors 101 are configured such that each of the sub-beams 14 (e.g., 14a-14d) provides illumination to a region of the sample plate.

[0047] According to an embodiment, the staggered mirrors 104 are positioned such that the mirror receiving the inner portion of the incident beam 13 guides the corresponding sub-beam 14 to a region located at the outer portion of the sample plate 106, and the mirror receiving the outer portion of the incident beam 13 guides the corresponding sub-beam 14 (e.g., 14a-14d) to the inner portion of the sample plate 106, thereby mitigating vignetting and / or attenuation effects of the imaging lens when capturing an image for the sample plate 106. In some embodiments, the staggered mirrors 104 are not in the same plane. The positioning of the staggered mirrors 104 (including the distance between the staggered mirrors 104) is determined by the positioning of regions of the sample plate 106.

[0048] In some embodiments, each of the staggered reflectors 101 can be of any shape, including circular, rectangular, square, sector, triangular, any irregular shape, or a combination thereof. In some aspects, the reflective surfaces of each of the staggered reflectors 104 (e.g., 104a-104d) have substantially the same shape as each region of the sample plate 106. The reflective surfaces of each of the staggered reflectors 104 (e.g., 104a-104d) may have substantially the same or larger dimensions than each region of the sample plate 106. In some embodiments, the sample plate 106 is disposed below a manifold 107. The manifold 107 is configured to secure the sample plate 106 by force and / or weight. In some aspects, the sample plate 106 includes a plurality of valves disposed along its edges and / or partitions, and the manifold 107 is in fluid communication with the valves. The manifold 107 does not cover any region of the sample plate 106 containing reaction sites. According to some embodiments, the incident angle of the reflective sub-beams 15 (e.g., 15a-15d) is determined to avoid or minimize the shadow cast by the manifold 107 on the sample plate 106. The manifold 107 may be made of plastic, metals including aluminum and steel, or combinations thereof.

[0049] In some embodiments, reference Figure 2A The sample plate 106 is substantially rectangular. The sample plate 106 may include four rectangular regions 301-304. Regions 301 and 304 are located at the outer portions of the sample plate 106. Regions 302 and 303 are located at the inner portions of the sample plate 106. The illumination system 100 may include staggered reflectors 104a-104d configured to split the incident beam 13 into sub-beams 14a-14d. Each sub-beam of sub-beams 14a-14d provides oblique illumination to each of regions 301-304 after being reflected by a guiding module 105 (not shown). According to some embodiments, such as... Figure 2AAs shown, sub-beams 14a and 14d, generated by staggered mirrors 104a and 104d reflecting the outer portion of the incident beam 13, are guided to regions 302 and 303, which are located within the inner portion of the sample plate 106. Sub-beams 14b and 14c, generated by staggered mirrors 104b and 104c reflecting the inner portion of the incident beam 13, are guided to regions 301 and 304, located on the outer portion of the sample plate 106. The staggered mirrors 104a-104d are configured to mitigate vignetting and / or attenuation effects in the image of the sample plate 106, thereby improving the image uniformity of the sample plate 106.

[0050] According to some embodiments, each region of sample plate 106 includes multiple reaction sites. Each reaction site may include a reaction mixture containing a biological sample. At least some of the reaction sites contain a dye capable of being excited by reflector beams 15 (e.g., 15a-15d). In some embodiments, the biological sample includes a nucleic acid sample. The nucleic acid sample may include ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA). In some embodiments, sample plate 106 includes a microfluidic chip. The microfluidic chip includes multiple microchambers. In some embodiments, the microfluidic chip includes multiple through-holes. In some cases, each reaction site is contained within a microchamber and / or through-hole of sample plate 106.

[0051] refer to Figure 1B As an alternative to the collimator 103 in the lighting system 100, which includes two cylindrical collimating lenses 103a and 103b, such as Figure 1BThe collimator 103 in the illustrated system 200 may include a single collimating lens. The single collimating lens has a focal length from 350 mm to 600 mm and all ranges and values ​​therebetween, including the ranges of 350 mm to 375 mm, 375 mm to 400 mm, 400 mm to 425 mm, 425 mm to 450 mm, 450 mm to 475 mm, 475 mm to 500 mm, 500 mm to 525 mm, 525 mm to 550 mm, 550 mm to 575 mm, and 575 mm to 600 mm. A single collimating lens can have a lens diameter of 100 mm to 130 mm and all ranges and values ​​therebetween, including 100 mm to 102 mm, 102 mm to 104 mm, 104 mm to 106 mm, 106 mm to 108 mm, 108 mm to 110 mm, 110 mm to 112 mm, 112 mm to 114 mm, 114 mm to 116 mm, 116 mm to 118 mm, 118 mm to 120 mm, 120 mm to 122 mm, 122 mm to 124 mm, 124 mm to 126 mm, 126 mm to 128 mm, and 128 mm to 130 mm. In some embodiments, a single collimating lens has a wavelength range of 400 nm to 800 nm. In some aspects, a single collimating lens is made of a material including borosilicate, plastic, or a combination thereof. Figure 1B As shown, lighting system 200 includes all the components and features of lighting system 100, except for different configurations of collimator 103.

[0052] According to the embodiments, the optical efficiencies of the lighting systems 100 and 200 are each approximately 15% to 35%, and all ranges and values ​​therebetween, including the 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%.

[0053] In some embodiments, illumination systems 100 and 200 are configured for use in bioanalytical instruments. Illumination systems 100 and 200 are configured for polymerase chain reaction (PCR) assays. In some aspects, illumination systems 100 and 200 are used for digital PCR (dPCR) assays. In a dPCR assay or experiment, according to some embodiments, a diluted solution containing at least one target polynucleotide or nucleotide sequence is subdivided into multiple reaction sites, such that at least some of these reaction sites contain a molecule or sequence of the target nucleotide or do not contain the target nucleotide sequence. When the reaction sites are subsequently thermally cycled in a PCR protocol, procedure, assay, process, or experiment, reaction sites containing one or more molecules of the target nucleotide sequence are amplified significantly and produce a positive, detectable detection signal, while samples containing no target nucleotide sequence are not amplified and do not produce a detection signal, or produce a signal below a predetermined threshold or noise level. In some embodiments, using Poisson statistics, the number of target nucleotide sequences distributed in the original solution between the reaction sites can be correlated with the number of reaction sites that produce a positive detection signal. In some embodiments, the detected signal can be used to determine the number or range of target molecules contained in the original solution. For example, the detection system can be configured to distinguish between reaction sites containing one target molecule and reaction sites containing two or more target molecules. Alternatively, the detection system can be configured to distinguish between reaction sites containing an equal to or less than a predetermined number of target molecules and reaction sites containing a greater than a predetermined number. In some embodiments, the qPCR and dPCR procedures, assays, or protocols are performed using a single, identical apparatus, instrument, system, and method.

[0054] B. Bioanalytical Instruments

[0055] In this embodiment, a bioanalytical instrument with an illumination system is provided, which improves optical efficiency and reduces background signal compared to conventional bioanalytical instruments. The instrument includes illumination system 100 or illumination system 200 as described above. In some embodiments, the instrument includes a base configured to receive one or more sample plates containing one or more biological samples. The base may include components configured to control one or more sample plates (e.g., ...). Figure 2A and Figure 2B The sample block assembly (sample plate 106) shown is a temperature-controlled sample block assembly. In some aspects, the base includes a thermal cycler, and the sample block includes two or more sections with independent temperature control, such that one or more sample plates or areas of sample plates can be processed at different temperatures. The base is configured to provide or perform PCR assays.

[0056] In some cases, the sample block comprises four zones with independent temperature control. Each of the four zones of the sample block is capable of independently controlling the temperature of a zone of the sample plate (e.g., zones 301, 302, 303, or 304 of sample plate 106).

[0057] According to an embodiment, the base of the instrument for bioanalysis may include a heated or temperature-controlled cover disposed above 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 may help maintain optical access to the biological sample in the sample plate.

[0058] In some embodiments, the instrument for bioanalysis includes an optical sensor configured to receive emission from a biological sample from one or more sample plates in response to excitation light. Exemplary optical sensors may include complementary metal-oxide-semiconductor (CMOS) sensors, charge-coupled device (CCD) sensors, or combinations thereof. The instrument for bioanalysis includes one or more emission filters disposed between one or more sample plates and the optical sensor. The one or more emission filters are configured to, for example, prevent excitation light from being reflected or scattered to the optical sensor. In some embodiments, the emission filters are arranged in an emission filter wheel. In some embodiments, at least some of the emitted light includes fluorescence emission from at least some of the biological sample in response to excitation by the excitation light.

[0059] According to embodiments, an instrument for bioanalysis includes an imaging unit configured to capture images of one or more sample plates disposed on a base. In some embodiments, the imaging unit may include an optical sensor circuit board and one or more sensor lenses. According to embodiments, the instrument for bioanalysis may include one or more electronic processors configured to control, monitor, and / or receive data from the optical sensors. In some embodiments, the instrument is configured to perform dPCR assays, and data analysis is based on images of biological samples on one or more sample plates.

[0060] As part of this disclosure, specific examples are provided below. These examples are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art will readily recognize that parameters can be changed or modified to produce substantially the same results.

[0061] Example

[0062] (The impact of lighting system configuration on image uniformity)

[0063] The lighting system configuration was tested, particularly the positioning of the staggered reflectors of lighting system 100 (e.g., Figure 1A The effect of (as shown) on the image uniformity of the sample plate.

[0064] QuantStudio TM Absolute Q TM The MAP16 plate was used as the sample plate. An LED light source was used to provide a diverging source beam (source beam 11). Then, through... Figure 1B Collimating lenses 103a and 103b, as shown, collimate the source beam 11 to form a substantially collimated rectangular incident beam 13. Tests were conducted as follows... Figure 2A and Figure 2B Two configurations of staggered mirrors are shown to determine the effect of the staggered mirror configuration on image uniformity. Using... Figure 2A The staggered mirror configuration shown (configuration A) guides sub-beams 14a and 14d, split from the outer portion of the incident beam 13, to regions 303 and 302, respectively, both of which are located within the sample plate. Figure 2A The sample plate 106 is located in the sample plate. Sub-beams 14b and 14c, which are split from the inner portion of the incident beam 13, are directed to regions 304 and 301, respectively. Both regions are located on the outer portion of the sample plate 106 to provide oblique illumination.

[0065] use Figure 2B The staggered reflector configuration shown (configuration B) guides sub-beams 14a' and 14d', split from the outer portion of the incident beam 13, to regions 301 and 304, respectively, both located on the outer portion of the sample plate 106. Sub-beams 14b' and 14c', split from the inner portion of the incident beam 13, are guided to regions 302 and 303, respectively, both located on the inner portion of the sample plate 106 to provide oblique illumination. FAMs are loaded respectively. TM Dyes and VIC ® Images of the dye sample plate were acquired using an imaging unit equipped with a CMOS sensor.

[0066] The result is Figures 3A to 3D It is displayed in the middle. Figure 3A The image shows an FAM with staggered mirrors in configuration B. TM The image obtained by dye loading the sample plate indicates significant image non-uniformity across the sample plate. Figure 3B The image shows an FAM using staggered mirrors in configuration A. TM The image acquired from the dye-loaded sample plate indicates improved image uniformity achieved by compensating for vignetting and / or attenuation effects from the imaging lens. Figure 3C The staggered mirrors of configuration B are shown for VIC. ® The image obtained by dye loading the sample plate indicates significant non-uniformity across the sample plate. Figure 3DThe image shows an interlaced mirror configuration A for VIC. ® Images acquired from a dye-loaded sample plate indicate improved image uniformity achieved by compensating for vignetting and / or attenuation effects from the imaging lens. The results demonstrate that image uniformity can be improved by mitigating vignetting and / or attenuation effects from the imaging lens through the positioning of staggered mirrors in the disclosed illumination system, by guiding the inner portion of the beam to the outer portion of the sample plate and vice versa.

[0067] In the context of this specification, at least the following embodiments are described. Embodiment 1 is an illumination system comprising: two or more staggered reflectors configured to split an incident light beam into at least two sub-beams; and a guiding module configured to guide the sub-beams to provide oblique illumination to a region. Embodiment 2 is the illumination system according to Embodiment 1, wherein the guiding module comprises one or more front reflectors configured to reflect the sub-beams such that the sub-beams have an angle of incidence greater than 0 degrees relative to the region. Embodiment 3 is the illumination system according to any one of Embodiments 1 and 2, wherein the staggered reflectors are positioned such that the incident light beam has an angle of incidence of approximately 45 degrees. Embodiment 4 is the illumination system according to any one of Embodiments 1 to 3, wherein the region includes a sample container. Embodiment 5 is the illumination system according to Embodiment 4, wherein the sample container includes a sample plate containing a plurality of reaction sites comprising a biological reaction mixture. Embodiment 6 is the illumination system according to Embodiment 5, wherein the sample plate includes a microfluidic chip comprising a plurality of microchambers configured to accommodate reaction sites. Example 7 is an illumination system according to any one of Examples 5 and 6, wherein a sub-beam providing oblique illumination to a region is capable of exciting a biological sample at a reaction site to generate emitted light. Example 8 is an illumination system according to Example 7, further comprising an optical sensor configured to receive emitted light from a sample plate. Example 9 is an illumination system according to any one of Examples 5 to 8, wherein a sample plate is disposed below a manifold, wherein the manifold is configured to fix the position of the sample plate. Example 10 is an illumination system according to any one of Examples 9, wherein the manifold is configured to provide a gas exchange mechanism for the sample plate. Example 11 is an illumination system according to any one of Examples 5 to 10, wherein the sample plate comprises at least two zones, each of two adjacent zones being separated by a separator. Example 12 is an illumination system according to Example 11, wherein each of at least two zones of the sample plate receives at least one sub-beam from staggered reflectors. Example 13 is an illumination system according to Examples 11 and 12, wherein the separated regions do not substantially directly receive illumination from the sub-beams. Example 14 is an illumination system according to any one of Examples 6 to 13, wherein the illumination efficiency of the illumination system is 15% to 30%. Example 15 is an illumination system according to any one of Examples 1 to 14, further comprising a collimator configured to collimate a narrowed beam to generate an incident beam. Example 16 is an illumination system according to Example 15, wherein the collimator comprises at least two cylindrical collimating lenses with different focal lengths, the at least two cylindrical collimating lenses being configured to generate an incident beam. Example 17 is an illumination system according to Example 15, wherein the collimator comprises a single collimating lens.Example 18 is a lighting system according to any one of Examples 15 to 17, further comprising a beam shaper configured to shape the source beam to produce a narrowed beam. Example 19 is a lighting system according to Example 18, wherein the source beam comprises a diverging LED beam, a laser beam, a xenon beam, a laser beam, an argon beam, a krypton beam, a tungsten halide beam, an incandescent beam, or a beam from any other type of commercially available light source. Example 20 is a lighting system according to Example 19, wherein the source beam is generated by an LED source, and the distance from the LED source to the tilted lighting area does not exceed 1200 mm. Example 21 is a lighting system according to any one of Examples 20, wherein the optical path from the LED source to the tilted lighting area is 600 mm to 1200 mm. Example 22 is a lighting system according to any one of Examples 1 to 21, wherein staggered reflectors are configured to split the incident beam with substantially no beam loss. Example 23 is an illumination system according to any one of Examples 1 to 22, wherein the illumination system is configured to provide illumination and / or excitation light for digital polymerase chain reaction (dPCR) assays.

[0068] Example 24 is an illumination system comprising: n staggered reflectors configured to split an incident light beam into m sub-beams; a guiding module configured to guide the sub-beams to provide oblique illumination to a sample plate, wherein the sample plate comprises k regions, each region comprising multiple reaction sites; and wherein n, m, and k are each integer, and m ≥ k, 2 ≤ m ≤ n. Example 25 is the illumination system according to Example 24, wherein the reaction sites comprise a reaction mixture containing a biological sample. Example 26 is the illumination system according to Example 25, wherein the sub-beams are capable of exciting the biological sample to generate emitted light. Example 27 is the illumination system according to Example 26, wherein the sub-beams providing oblique illumination to the sample plate are not parallel to the emitted light. Example 28 is the illumination system according to Example 27, wherein the sub-beams providing oblique illumination to the sample plate have an incident angle of 25 to 40 degrees. Example 29 is the illumination system according to Example 27, wherein the sub-beams providing oblique illumination to the sample plate have an incident angle of approximately 32 degrees. Example 30 is an illumination system according to any one of Examples 24 to 29, wherein the sample plate includes a microfluidic chip. Example 31 is an illumination system according to Example 30, wherein the microfluidic chip includes a plurality of microchambers. Example 32 is an illumination system according to Example 31, wherein at least some of the microchambers each contain a reaction site. Example 33 is an illumination system according to any one of Examples 31 and 32, wherein the sample plate includes at least 20,000 reaction sites. Example 34 is an illumination system according to any one of Examples 24 to 33, wherein the sample plate includes a plurality of regions, each region including a plurality of reaction sites. Example 35 is an illumination system according to Example 34, wherein adjacent regions are separated by separators, and the separators substantially do not receive illumination from sub-beams. Example 36 is an illumination system according to Example 35, wherein staggered mirrors are configured such that each sub-beam provides illumination to one region. Example 37 is an illumination system according to Example 36, wherein staggered mirrors are configured such that a mirror receiving an inner portion of an incident beam guides a corresponding sub-beam to a region located at an outer portion of the sample plate. Example 38 is an illumination system according to Example 37, wherein staggered mirrors are arranged to compensate for vignetting and / or attenuation effects of the imaging lens when acquiring an image of the sample plate, thereby improving image uniformity of the sample plate. Example 39 is an illumination system according to any one of Examples 35 to 38, wherein the staggered mirrors are not in the same plane, and the distance between the staggered mirrors is determined by the positioning of a region of the sample plate. Example 40 is an illumination system according to any one of Examples 24 to 39, further comprising a beam shaper configured to shape a source beam to produce a narrowed beam; and one or more collimators configured to collimate the narrowed beam to produce an incident beam.Example 41 is an illumination system according to Example 40, wherein the beam shaper is a beam shaper lens. Example 42 is an illumination system according to any one of Examples 40 and 41, wherein the collimator includes a collimating lens. Example 43 is an illumination system according to any one of Examples 40 and 41, wherein the collimator includes at least two cylindrical collimating lenses with different focal lengths, the at least two cylindrical collimating lenses being configured to generate a rectangular incident beam. Example 44 is an illumination system according to any one of Examples 40 to 43, wherein tilted illumination of the sample plate can improve the optical efficiency of the illumination system compared to coaxial illumination. Example 45 is an illumination system according to any one of Examples 40 to 44, further comprising a light source configured to provide a source beam. Example 46 is an illumination system according to Example 45, wherein the light source includes an LED light source, a laser light source, a halogen light source, a tungsten light source, a xenon lamp light source, an argon lamp light source, a krypton lamp light source, an incandescent light source, or a combination thereof. Example 47 is an illumination system according to any one of Examples 45 and 46, wherein the optical path from the light source to the sample plate is approximately 600 mm to 1200 mm. Example 48 is an illumination system according to any one of Examples 24 to 47, wherein the optical efficiency of the illumination system is approximately 15% to 35%. Example 49 is an illumination system according to any one of Examples 24 to 48, wherein the guiding module includes one or more front reflectors configured to reflect a sub-beam onto the sample plate at an incident angle of 25 degrees to 40 degrees. Example 50 is an illumination system according to Example 49, wherein the guiding module includes a single front reflector. Example 51 is an illumination system according to Example 49, wherein the guiding module includes... n Each front reflector corresponds to an interleaved reflector. Example 52 is an illumination system according to any one of Examples 24 to 51, wherein the illumination system is an illumination system for a digital PCR system.

[0069] Example 53 is an instrument for bioanalysis, comprising: a base configured to receive one or more sample plates containing biological samples; and an illumination system configured to illuminate the one or more sample plates. The illumination system includes: n A series of interlaced reflectors are configured to split the incident light beam into... m Sub-beams; a front reflector configured to guide the sub-beams to provide oblique illumination to one or more sample plates and / or excite biological samples to generate emitted light, wherein the incident beam and the sub-beams are collimated beams; wherein the sample plates include kEach region includes multiple reaction sites, where n, m, and k are each integers, and n ≥ k, 2 ≤ m ≤ n. The instrument includes an optical sensor configured to receive emitted light from a biological sample. Example 54 is an instrument according to Example 53, wherein the base further includes a thermal cycler configured to perform a polymerase chain reaction on the biological sample. Example 55 is an instrument according to any one of Examples 53 and 54, wherein the instrument is configured to perform digital PCR assays. Example 56 is an instrument according to any one of Examples 53 to 55, wherein the sample plate is a microfluidic chip comprising at least 20,000 partitions. Example 57 is an instrument according to Example 56, wherein the microfluidic chip comprises at least 20,000 microchambers. Example 58 is an instrument according to any one of Examples 53 to 57, further comprising one or more emission filters disposed between one or more sample plates and an optical sensor. Example 59 is the instrument according to Example 58, wherein at least some of the emitted light comprises fluorescence emission from at least some of the biological sample in response to excitation by excitation light. Example 60 is the instrument according to Example 59, further comprising an imaging unit configured to capture an image of a sample plate. Example 61 is the instrument according to any one of Examples 48 to 55, wherein the illumination system further comprises: a light source configured to provide a source beam; a beam shaper configured to shape the source beam to produce a narrowed beam; and one or more collimators configured to collimate the narrowed beam to produce an incident beam. Example 62 is the instrument according to Example 56, wherein the optical path from the light source to one or more sample plates is approximately 600 mm to 1200 mm. Example 63 is the instrument according to any one of Examples 48 to 57, wherein the optical sensor comprises a complementary metal-oxide-semiconductor sensor.

[0070] While embodiments of the present application and their advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the embodiments as defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to the specific implementations of the systems, instruments, machines, articles, compositions of matter, methods, and / or steps described in the specification. As will be readily apparent to those skilled in the art, based on the foregoing disclosure, currently existing or later-developed systems, instruments, processes, machines, articles, compositions of matter, methods, or steps that perform substantially the same functions or achieve substantially the same results as the corresponding embodiments described herein can be utilized. Therefore, the appended claims are intended to include such processes, machines, articles, compositions of matter, methods, or steps within their scope.

Claims

1. A lighting system comprising: Two or more staggered reflectors are configured to split an incident beam into at least two sub-beams; and A guiding module is configured to guide the sub-beam to provide oblique illumination to the area.

2. The lighting system of claim 1, wherein the guiding module includes one or more front reflectors configured to reflect the sub-beam such that the sub-beam has an angle of incidence greater than 0 degrees relative to the region.

3. The lighting system according to any one of claims 1 and 2, wherein the staggered reflectors are positioned such that the incident light beam has an incident angle of about 45 degrees.

4. The lighting system according to any one of claims 1 to 3, wherein the area includes a sample holder.

5. The lighting system of claim 4, wherein the sample holder comprises a sample plate containing a plurality of reaction sites comprising a biological reaction mixture.

6. The lighting system of claim 5, wherein the sample plate comprises a microfluidic chip, the microfluidic chip comprising a plurality of microchambers configured to accommodate the reaction sites.

7. The illumination system according to any one of claims 5 and 6, wherein the sub-beam providing the tilted illumination to the sample plate is capable of exciting the biological sample at the reaction site to generate emitted light.

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

9. The lighting system according to any one of claims 5 to 8, wherein the sample plate is disposed below the manifold, wherein the manifold is configured to fix the position of the sample plate.

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

11. The lighting system according to any one of claims 5 to 10, wherein the sample plate comprises at least two zones, each of the two adjacent zones being separated by a partition region.

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 reflectors.

13. The lighting system according to any one of claims 11 and 12, wherein the partitioned area does not substantially directly receive illumination from the sub-beam.

14. The lighting system according to any one of claims 6 to 13, wherein the lighting efficiency of the lighting system is 15% to 30%.

15. The lighting system according to any one of claims 1 to 14, further comprising: A collimator is configured to collimate the narrowed beam to produce the incident beam.

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

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

18. The lighting system according to any one of claims 15 to 17, further comprising: A beam shaper configured to shape the source beam to produce the narrowed beam.

19. The lighting system of claim 18, wherein the source beam comprises a diverging LED beam, a laser beam, a xenon beam, a laser beam, an argon beam, a krypton beam, a tungsten halide beam, an incandescent beam, or a combination thereof.

20. The lighting system of claim 19, wherein the source beam is generated by an LED source, and the distance from the LED source to the oblique lighting area does not exceed 1200 mm.

21. The lighting system according to any one of claims 20, wherein the optical path from the LED source to the tilted lighting area is 600 mm to 1200 mm.

22. The lighting system according to any one of claims 1 to 21, wherein the staggered reflectors are configured to split the incident beam with substantially no beam loss.

23. The illumination system according to any one of claims 1 to 22, wherein the illumination system is configured to provide illumination and / or excitation light for digital polymerase chain reaction (dPCR) assays.

24. A lighting system comprising: n staggered mirrors are configured to split the incident beam into m sub-beams; A guiding module configured to guide the sub-beam to provide tilted illumination to the sample plate; The sample plate comprises k regions, each region including multiple reaction sites; and Where n, m and k are each integers, and m ≥ k, 2≤m≤n.

25. The lighting system of claim 24, wherein the reaction site comprises a reaction mixture containing a biological sample.

26. The illumination system of claim 25, wherein the sub-beam is capable of exciting the biological sample to generate emitted light.

27. The illumination system of claim 26, wherein the sub-beam providing oblique illumination to the sample plate is not parallel to the emitted light.

28. The illumination system of claim 27, wherein the sub-beam providing oblique illumination to the sample plate has an incident angle of 25 to 40 degrees.

29. The illumination system of claim 27, wherein the sub-beam providing oblique illumination to the sample plate has an incident angle of approximately 32 degrees.

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

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

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

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

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

35. The lighting system of claim 34, wherein two adjacent areas are separated by a separator, and the separator substantially does not receive illumination from the sub-beam.

36. The lighting system of claim 35, wherein the staggered reflectors are configured such that each sub-beam provides illumination to a region.

37. The illumination system of claim 36, wherein the staggered reflectors are configured such that the reflector receiving the inner portion of the incident light beam guides the corresponding sub-beam to an area located at the outer portion of the sample plate.

38. The illumination system of claim 37, wherein the staggered reflectors are arranged to compensate for vignetting and / or attenuation effects of the imaging lens when acquiring an image of the sample plate, thereby improving the image uniformity of the sample plate.

39. The lighting system according to any one of claims 35 to 38, wherein the staggered reflectors are not in the same plane, and the distance between the staggered reflectors is determined by the positioning of the area of ​​the sample plate.

40. The lighting system according to any one of claims 24 to 39, further comprising: A beam shaper configured to shape a source beam to produce a narrowed beam; and One or more collimators are configured to collimate the narrowed beam to produce the incident beam.

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

42. The lighting system according to any one of claims 40 and 41, wherein the collimator comprises a collimating lens.

43. The illumination system according to any one of claims 40 and 41, wherein the collimator comprises at least two cylindrical collimating lenses having different focal lengths, the at least two cylindrical collimating lenses being configured to generate a rectangular incident beam.

44. The illumination system according to any one of claims 40 to 43, wherein the tilted illumination of the sample plate is configured to improve the optical efficiency of the illumination system compared to coaxial illumination.

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

46. ​​The lighting 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 lighting system according to any one of claims 45 and 46, wherein the optical path from the light source to the sample plate is about 600 mm to 1200 mm.

48. The lighting system according to any one of claims 24 to 47, wherein the optical efficiency of the lighting system is about 15% to 35%.

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

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

51. The lighting system of claim 49, wherein the guiding module comprises n front reflectors, and each front reflector corresponds to an interleaved reflector.

52. The lighting system according to any one of claims 24 to 51, wherein the lighting system is a lighting system for a digital PCR system.

53. An instrument for biological analysis, said instrument comprising: A base, configured to receive one or more sample plates containing biological samples; An illumination system configured to illuminate the one or more sample plates, wherein the illumination system includes: n staggered mirrors are configured to split the incident beam into m sub-beams; A front reflector is configured to guide the sub-beam to provide oblique illumination to the one or more sample plates and / or excite the biological sample to generate an emitted beam, wherein the incident beam and the sub-beam are substantially collimated beams; The sample plate comprises k regions, each region including multiple reaction sites, where n, m, and k are integers, and m ≥ k, 2 ≤ m ≤ n; and An optical sensor is configured to receive emitted light from the biological sample.

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

55. The instrument according to any one of claims 53 and 54, wherein the instrument is configured to perform digital PCR assays.

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

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

58. The instrument according to any one 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 emitted light comprises fluorescence emission from at least some of the biological sample in response to excitation by the excitation light.

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

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

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

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

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