US-12625076-B2 - Fluorescence detection system

Abstract

A fluorescence detection system, including apparatus and methods, suitable for qPCR and other fluorescence-based analyses. The system may comprise various components, including a stage, an illumination module, a detection module, and an optical relay structure. The stage may be configured to support a sample holder. The illumination module may include one or more discrete light sources configured to produce excitation light. The detection module may be configured to detect fluorescence emission light produced, in response to the excitation light, by a fluorescent sample positioned in the sample holder. The optical relay structure may include a beamsplitter assembly configured to direct the excitation light from the illumination module along an illumination path to the sample holder and to direct the fluorescence emission light from the sample holder along a response path to the imaging module. The system may enhance the quality of excitation light hitting samples in the sample holder.

Inventors

• Evan Thrush
• Stephen L. Swihart
• Eli A. Hefner
• Michael Griffin
• Li Lu

Assignees

• BIO-RAD LABORATORIES, INC.

Dates

Publication Date

20260512

Application Date

20221103

Claims (16)

1. 1 . A fluorescence detection system, comprising: a stage configured to support a sample holder; an illumination module including a plurality of discrete light sources, each light source configured to produce spectrally distinct excitation light; a detection module configured to detect fluorescence emission light produced, in response to the excitation light from at least one of the light sources, by a fluorescent sample positioned in the sample holder; and an optical relay structure including a beamsplitter assembly configured to direct the excitation light from the illumination module along an illumination path to the sample holder and to direct the fluorescence emission light from the sample holder along a response path to the detection module, wherein the illumination path and the response path overlap between the beamsplitter assembly and the sample holder; wherein: the optical relay structure includes a plurality of beamsplitters corresponding in number to the plurality of light sources, each light source is optically coupled with a unique one of the beamsplitters, given pairs of a light source and a beamsplitter can alternately be positioned to illuminate the sample, each pair of an optically coupled light source and a beamsplitter is mounted on an excitation wheel such that given pairs can alternately be positioned to illuminate the sample by rotating the wheel, the optical relay structure further includes a plurality of emission filters corresponding in number to the number of optically coupled pairs of a light source and a beamsplitter and configured to reduce the amount of excitation light that reaches the detection module, and the emission filters are mounted on an emission wheel that can be rotated in tandem with the excitation wheel.
2. 2 . The system of claim 1 , wherein the excitation wheel and emission wheel are coaxial and their rotation is driven by a common driver.
3. 3 . The system of claim 1 , wherein power for the light sources is derived through wires connecting the light sources to a shaft about which the excitation wheel rotates.
4. 4 . The system of claim 1 , wherein the optical relay structure further includes a condenser lens positioned in the illumination path such that excitation light exiting the condenser lens and incident on the sample holder is collimated and perpendicular to a plane of the sample holder.
5. 5 . The system of claim 1 wherein the optical relay structure further includes a field lens positioned in the response path downstream from the beamsplitter assembly and configured to collect fluorescence emission light and direct it toward the detector module.
6. 6 . The system of claim 5 , wherein the field lens is positioned upstream from one of the emission filters.
7. 7 . The system of claim 1 , further comprising a controller configured to manage at least one of the stage, the illumination module, the detection module, and the optical relay structure.
8. 8 . The system of claim 1 , wherein the stage further includes a heating block configured to cycle the temperature of the sample such that PCR occurs, altering the fluorescence of the sample.
9. 9 . The system of claim 1 , wherein the light sources are LEDs.
10. 10 . A method of reading fluorescence, comprising: providing the fluorescence detection system of claim 1 ; providing a sample holder having a plurality of sample sites, each sample site containing a fluorescent sample; positioning a first one of the light sources and a first one of the beamsplitters so that the sample holder is illuminated with light from the first light source, and collecting a first image of fluorescence; and positioning a second one of the light sources and a second one of the beamsplitters such that the sample holder is illuminated with light from the second light source, and collecting a second image of fluorescence from the fluorescent samples.
11. 11 . A fluorescence detection system, comprising: a stage configured to support a sample holder; an illumination module including a plurality of discrete light sources, each light source configured to produce spectrally distinct excitation light; a detection module configured to detect fluorescence emission light produced, in response to the excitation light from at least one of the light sources, by a fluorescent sample positioned in the sample holder; and an optical relay structure including a beamsplitter assembly configured to direct the excitation light from the illumination module along an illumination path to the sample holder and to direct the fluorescence emission light from the sample holder along a response path to the detection module, wherein the illumination path and the response path overlap between the beamsplitter assembly and the sample holder; wherein the beamsplitter assembly includes a plurality of beamsplitters, each beamsplitter forming a pair with one of the light sources, each of the pairs of light source and beamsplitter being mounted on a rotatable wheel for alternately positioning a selected light source and beam splitter pair to illuminate a sample held by the sample holder.
12. 12 . The system of claim 11 , wherein the optical relay structure further includes an emission filter positioned in the response path between the beamsplitter and the detection module and configured to reduce the amount of excitation light that reaches the detection module.
13. 13 . The system of claim 12 , wherein there is a distinct emission filter for each light source, and wherein the distinct emission filters can alternately be placed in the response path depending on which light source is being used to produce excitation light.
14. 14 . The system of claim 11 , wherein the optical relay structure further includes a field lens positioned in the response path downstream from a selected beamsplitter and configured to collect fluorescence emission light and direct it toward the detection module.
15. 15 . The system of claim 11 , further comprising a controller configured to manage at least one of the stage, the illumination module, the detection module, and the optical relay structure.
16. 16 . The system of claim 11 , wherein the stage further includes a heating block configured to cycle the temperature of the sample such that PCR occurs, altering the fluorescence of the sample.

Description

CROSS-REFERENCE TO PRIORITY APPLICATION This application is a continuation of PCT Patent Application Serial No. PCT/US2022/047322, filed Oct. 20, 2022, which in turn is based upon and claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 63/257,973, filed Oct. 20, 2021. Each of these applications is incorporated herein by reference in its entirety for all purposes. INTRODUCTION Fluorescence is an optical phenomenon involving the absorption and reemission of light. It has become the basis of many analytical and diagnostic techniques in biology and medicine due to its sensitivity, selectivity, and versatility. These techniques include polymerase chain reaction (PCR), including real-time or quantitative PCR (qPCR), among others. In a fluorescence-based PCR assay, such as qPCR, one or more fluorescent compounds, termed “fluorophores,” are used to assess the status of a PCR reaction. The PCR reaction, in turn, may be used to determine gene expression and the presence or absence of a pathogen or pathogenic state, among many other uses. PCR is used to amplify nucleic acids. Fluorescence is used in PCR to detect the amplified nucleic acids, typically by nonspecific binding to any amplified product or specific binding to a particular amplified product. The binding, which is indicative of the reaction, is associated with a measurable change in fluorescence. Fluorescence-based PCR assays may be conducted on a single sample or many samples by shining “excitation” light on those samples and observing the resulting fluorescence “emission” light. It is important in these fluorescence-based analyses to have quality illumination, free from spatial inhomogeneities and shadows. Otherwise, variations in fluorescence may reflect variations in illumination rather than variations in the samples. Unfortunately, current systems frequently use off-axis illumination than can lead to gradients in excitation light and shadows when samples are contained in wells. Thus, there is a need for improved fluorescence detection systems for use in PCR and other fluorescence-based techniques. SUMMARY The present disclosure provides a fluorescence detection system, including apparatus and methods, suitable for qPCR and other fluorescence-based analyses. The system may comprise various components, including a stage, an illumination module, a detection module, and an optical relay structure. The stage may be configured to support a sample holder. The illumination module may include one or more discrete light sources configured to produce excitation light. The detection module may be configured to detect fluorescence emission light produced, in response to the excitation light, by a fluorescent sample positioned in the sample holder. The optical relay structure may include a beamsplitter assembly configured to direct the excitation light from the illumination module along an illumination path to the sample holder and to direct the fluorescence emission light from the sample holder along a response path to the imaging module. The system may enhance the quality of excitation light hitting samples in the sample holder, for example, by collimating and/or homogenizing the light. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a high-level schematic view of an exemplary fluorescence detection system, showing (A) a stage supporting a sample holder that, in turn, supports a fluorescent sample, (B) an illumination module including at least one light source configured to produce excitation light, (C) a detection module configured to detect fluorescence emission light produced by the sample in response to the excitation light, and (D) an optical relay structure including a beamsplitter assembly configured to direct the excitation light from the illumination module along an illumination path to the sample and to direct the fluorescence emission light from the sample along a response path to the detection module. FIG. 2 is a schematic view of a first embodiment of the fluorescence detection system of FIG. 1, showing details of a first exemplary optical relay structure, including a condenser lens positioned in the illumination path one focal length from the light source and upstream from the beamsplitter, such that excitation light produced by the light source uniformly illuminates the sample, and a field lens positioned in the response path downstream from the beamsplitter to collect and focus fluorescence emission light onto the detection module. FIG. 3 is a schematic view of portions of the embodiment of FIG. 2, showing how a tiltable beamsplitter allows both on- and off-axis light sources to deliver uniform illumination to the sample. FIG. 4 is a schematic view of a second embodiment of the fluorescence detection system of FIG. 1, showing details of a second exemplary optical relay structure, including a condenser lens positioned in the illumination path one focal length from the light source and downstream from the beamsplitte