Systems and methods for evaluating biological samples - Patents.com

Abstract

To provide systems and methods for assessing biological samples.SOLUTION: An instrument for biological analysis includes a base member, an excitation source, an optical sensor, an excitation optical system, and an emission optical system. The base member is configured to receive a sample holder comprising a plurality of biological samples. The optical sensor is configured to receive emissions from the biological samples in response to the excitation source. The instrument may additionally include a sensor lens enclosed by a lens case, and a focusing mechanism including a gear that engages the lens case, the focusing mechanism being accessible from outside the enclosure for adjusting a focus. The instrument may further include a sensor aperture disposed along an emission optical path, and a blocking structure disposed to cooperate with the sensor aperture such that reflected radiation from an illuminated surface near the sample holder is not received by the optical sensor and also not reflected off another surface of the instrument.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] The present invention relates generally to systems, devices and methods for observing, testing and / or analyzing one or more biological samples, and more particularly to systems, devices and methods that include optical systems for observing, testing and / or analyzing one or more biological samples. Summary of the Invention [Means for solving the problem]

[0002] Although the devices, apparatus, systems and methods according to the embodiments of the present invention generally relate to dPCR and qPCR, the embodiments of the present invention may be applicable to any PCR process, experiment, assay or protocol in which a large number of reaction regions are processed, observed and / or measured. In a dPCR assay or experiment according to the embodiments of the present invention, a dilute solution containing at least one target polynucleotide or nucleotide sequence is divided into a plurality of reaction regions, so that at least some of these reaction regions contain one molecule of the target nucleotide sequence or none of the target nucleotide sequence. When the reaction regions are then thermally cycled in a PCR protocol, procedure, assay, process or experiment, the reaction regions containing one or more molecules of the target nucleotide sequence are highly amplified and generate a positive detectable detection signal, while the reaction regions that do not contain the target nucleotide sequence are not amplified and generate no detection signal or generate a signal or noise level below a predetermined threshold. Using Poisson statistics, the number of target nucleotide sequences in the original solution distributed among the reaction regions will correlate with the number of reaction regions that generate a positive detection signal. In some embodiments, the detected signal may be used to determine the number or range of numbers of target molecules contained in the original solution. For example, the detection system may be configured to distinguish between reaction areas containing one target molecule and reaction areas containing two or at least two target molecules.In addition, or alternatively, the detection system may be configured to distinguish between reaction areas containing a predetermined amount of target molecules or a number of target molecules less than a predetermined amount and reaction areas containing a number of target molecules greater than the predetermined amount.In certain embodiments, the process, assay or protocol for both qPCR and dPCR is carried out using one and the same device, apparatus or system and method. The present invention provides, for example, the following: (Item 1) 1. An apparatus for biological analysis comprising: an excitation source; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path; an emission optical system disposed along an emission light path; and an imaging unit, the imaging unit comprising: a first surface and an opposing second surface; Sensor lens and; a lens case at least partially enclosing the lens sensor; a light collecting mechanism having a gear configured to engage the lens case; the first surface comprises a surface of the sensor lens; the second surface having an optical sensor circuit board; 13. An apparatus for biological analysis, wherein the light collection mechanism is configured to be accessible from outside an enclosure for adjusting the focus of the sensor lens. (Item 2) 1. An apparatus for biological analysis comprising: an excitation source; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along the excitation optical path; an emission optical system disposed along an emission light path; a sensor lens configured to direct luminescence from at least a portion of the biological sample towards the optical sensor; an illuminated surface disposed along the excitation light path, the illuminated surface configured to generate reflected radiation including radiation from the excitation source reflected by the illuminated surface; and a radiation shield, the radiation shield comprising: a sensor aperture disposed along the emitted light path; and a blocking structure positioned to cooperate with the sensor opening such that the reflected radiation is not received by the optical sensor and is not reflected onto another surface of the device. (Item 3) 1. An apparatus for biological analysis comprising: an excitation source; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along the excitation optical path; an emission optical system disposed along an emission light path; and an energy or force detection unit, the energy or force detection unit comprising: an energy or force sensor disposed outside the optical path; a light pipe disposed adjacent to the beam splitter and configured to transfer radiation from the beam splitter to the force sensor. (Item 4) 1. An apparatus for biological analysis comprising: an excitation source; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along the excitation optical path; an emission optical system disposed along an emission light path; a position light source configured to emit radiation and a corresponding position sensor configured to receive radiation from the position light source, the position light source and the position sensor configured to generate a position signal indicative of a position of an optical element disposed along at least one of the optical paths; and a radiation shield configured to at least partially block radiation from the positional light source. (Item 5) 1. An apparatus for biological analysis comprising: an excitation source; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along the excitation optical path; an emission optical system disposed along an emission light path; an optical enclosure enclosing the optical path, the optical enclosure including a split wire grommet configured to pass a wire or cable from a location outside the enclosure to a location inside the enclosure while blocking light outside the enclosure from entering the enclosure; and a lens hole cover configured to provide three-dimensional adjustment of a sensor lens while blocking light outside the enclosure from entering the enclosure. (Item 6) 1. An apparatus for biological analysis comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an optical sensor configured to receive luminescence from the biological sample in response to an excitation source; an excitation optical system disposed along the excitation optical path; an emission optical system disposed along the emission light path; An apparatus for biological analysis, wherein the optical sensor is a complementary metal oxide semiconductor sensor. (Item 7) 7. The apparatus of claim 1, further comprising a beam splitter disposed along both the excitation light path and the emission light path. (Item 8) 1. An apparatus for biological analysis comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples, the base member including a thermal cycler configured to perform a polymerase chain reaction assay on the divided biological samples; an excitation source configured to generate a first excitation beam characterized by a first wavelength and a second excitation beam characterized by a second wavelength different from the first wavelength; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system including a sample lens disposed along an excitation optical path between the excitation source and the base member and configured to direct the excitation beam toward the sample holder; an emission optical system disposed along an emission optical path between the base member and the optical sensor and configured to direct emission light from the biological sample to the optical sensor; a beam splitter disposed along both the excitation light path and the emission light path, the beam splitter being positioned to accept a first excitation beam and to accept a second excitation beam, the sample lens being positioned along the excitation light path between the beam splitter and the base member; a beam dump that receives the excitation beam radiation from the beam splitter, where less than 10% of the excitation beam radiation is reflected back to the beam splitter; An imaging unit, the imaging unit comprising: a first surface and an opposing second surface; Sensor lens and; a lens case at least partially enclosing the lens sensor; a light collecting mechanism having a gear configured to engage the lens case; the first surface comprises a surface of the sensor lens; the second surface having an optical sensor circuit board; an imaging unit, the focusing mechanism configured to be accessible from outside an enclosure for adjusting the focus of the sensor lens; an illuminated surface disposed along the excitation light path between the beam splitter and the base member, the illuminated surface configured to generate reflected radiation, the reflected radiation including radiation from the excitation source reflected by the illuminated surface; A radiation field, the radiation field comprising: a sensor aperture disposed along the emitted light path between the beam splitter and the sensor lens; a blocking structure arranged to cooperate with the sensor opening such that the reflected radiation is not received by the optical sensor and is not reflected onto another surface of the device; An energy or force detection unit, the energy or force detection unit comprising: an energy or force sensor disposed outside the optical path; an energy or force detection unit including a light pipe disposed adjacent to the beam splitter and configured to transfer radiation from the beam splitter to the force sensor described above; a position light source configured to emit radiation and a corresponding position sensor configured to receive radiation from the position light source, the position light source and the position sensor configured to generate a position signal indicative of a position of an optical element disposed along at least one optical path; a radiation shield configured to at least partially block radiation from the positional light source; an optical enclosure enclosing the optical path, the optical enclosure including a split wire grommet configured to pass a wire or cable from a location outside the enclosure to a location inside the enclosure while blocking light outside the enclosure from entering the enclosure; a lens hole cover configured to perform three-dimensional adjustment of a sensor lens while blocking light outside the enclosure from entering the enclosure; An apparatus for biological analysis, wherein the optical sensor is a complementary metal oxide semiconductor sensor. (Item 9) Item 9. The device of item 8, wherein the blocking structure is positioned to cooperate with the sensor opening such that the reflected radiation does not impinge on the sensor lens without also being reflected off another surface of the device. (Item 10) 1. An apparatus for biological analysis comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an excitation source configured to generate a first excitation beam characterized by a first wavelength and a second excitation beam characterized by a second wavelength different from the first wavelength; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path between the excitation source and the sample holder; an emission optical system disposed along an emission optical path between the base member and the optical sensor and configured to direct emission light from the biological sample to the optical sensor; and an imaging unit, the imaging unit comprising: a first surface and an opposing second surface; Sensor lens and; a lens case at least partially enclosing the lens sensor; a light collecting mechanism having a gear configured to engage the lens case; the first surface comprises a surface of the sensor lens; the second surface having an optical sensor circuit board; 13. An apparatus for biological analysis, wherein the light collection mechanism is configured to be accessible from outside an enclosure for adjusting the focus of the sensor lens. (Item 11) 1. An apparatus for biological analysis comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an excitation source configured to generate a first excitation beam characterized by a first wavelength and a second excitation beam characterized by a second wavelength different from the first wavelength; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path between the excitation source and the sample holder; an emission optical system disposed along an emission optical path between the sample holder and the optical sensor and configured to direct emission from the biological sample to the optical sensor; a sensor lens configured to direct luminescence from at least a portion of the biological sample to an optical sensor; an illuminated surface disposed along the excitation optical path between a beam splitter and the base member, the illuminated surface configured to generate reflected radiation including radiation from the excitation source reflected by the illuminated surface; and a radiation shield, the radiation shield comprising: a sensor aperture disposed along the emitted light path between the beam splitter and the sensor lens; and a blocking structure positioned to cooperate with the sensor opening such that reflected radiation is not received by the optical sensor and is not reflected onto another surface of the device. (Item 12) 1. An apparatus for biological analysis comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an excitation source configured to generate a first excitation beam characterized by a first wavelength and a second excitation beam characterized by a second wavelength different from the first wavelength; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path between the excitation source and the base member; an emission optical system disposed along an emission optical path between the base member and the optical sensor and configured to direct emission light from the biological sample to the optical sensor; and an energy or force detection unit, the energy or force detection unit comprising: an energy or force sensor disposed outside the optical path; and a light pipe disposed adjacent to the beam splitter and configured to transfer radiation from the beam splitter to the force sensor. 1. An apparatus for biological analysis comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an excitation source configured to generate a first excitation beam characterized by a first wavelength and a second excitation beam characterized by a second wavelength different from the first wavelength; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path between the origin and the base member; an emission optical system disposed along an emission optical path between the base member and the optical sensor and configured to direct emission light from the biological sample to the optical sensor; a beam splitter disposed along both the excitation light path and the emission light path, the beam splitter being positioned to accept the first excitation beam and to accept the second excitation beam, the sample lens being positioned along the excitation light path between the beam splitter and a base member; a position light source configured to emit radiation and a corresponding position sensor configured to receive radiation from the position light source, the position light source and the position sensor configured to generate a position signal indicative of a position of an optical element disposed along at least one optical path; and a radiation shield configured to at least partially block radiation from the positional light source. (Item 14) 1. An apparatus for biological analysis comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an excitation source configured to generate a first excitation beam characterized by a first wavelength and a second excitation beam characterized by a second wavelength different from the first wavelength; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path between the excitation source and the base member; an emission optical system disposed along an emission optical path between the base member and the optical sensor and configured to direct emission light from the biological sample to the optical sensor; a beam splitter disposed along both the excitation light path and the emission light path, the beam splitter positioned to accept the first excitation beam and to accept the second excitation beam; an optical enclosure including a split wire grommet configured to pass a wire or cable from a location outside the enclosure to a location inside the enclosure while enclosing an optical path and blocking light outside the enclosure from entering the enclosure; and a lens hole cover configured to provide three-dimensional adjustment of a sensor lens while blocking light outside the enclosure from entering the enclosure. (Item 15) 1. An apparatus for biological analysis comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an excitation source configured to generate a first excitation beam characterized by a first wavelength and a second excitation beam characterized by a second wavelength different from the first wavelength; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path between the excitation source and the base member; an emission optical system disposed along an emission optical path between the base member and the optical sensor and configured to direct emission light from the biological sample to the optical sensor; a beam splitter disposed along both the excitation light path and the emission light path, the beam splitter being positioned to accept the first excitation beam and to accept the second excitation beam; An apparatus for biological analysis, wherein the optical sensor is a complementary metal oxide semiconductor sensor. (Item 16) 16. The apparatus of items 7, 8, 10, 11, 12, 13, 14 or 15, further comprising one or more emission filters disposed along the emission light path. (Item 17) 16. The apparatus of claim 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein at least a portion of the emitted light comprises fluorescent emission from at least a portion of the biological sample in response to at least one of the excitation beams. (Item 18) 16. The apparatus of claim 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein at least a portion of the emitted light comprises fluorescent emission from at least a portion of the biological sample in response to at least one of the excitation beams. (Item 19) 16. The apparatus of claim 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein at least a portion of the emitted light comprises radiation from at least one of the excitation beams that is reflected, refracted, diffracted, scattered, or polarized by at least some of the biological sample. (Item 20) 16. The apparatus of claim 1, further comprising a temperature-controlled cover disposed along the excitation light path between the base member and the beam splitter. (Item 21) 21. The apparatus of claim 20, further comprising a mirror disposed along the excitation light path between the base member and the beam splitter. (Item 22) 16. The apparatus of claim 7, 8, 11, 12, 13, 14 or 15, further comprising a mirror disposed along the excitation light path between the base member and the beam splitter. (Item 23) 16. The apparatus of claim 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the base member comprises a sample block assembly configured to control the temperature of the sample holder or the biological sample. (Item 24) 24. The apparatus of claim 23, wherein the sample block assembly comprises one or more of a sample block, a Peltier device, or a heat sink. (Item 25) 16. The apparatus of claim 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14 or 15, wherein the base member comprises a thermal controller. (Item 26) 26. The apparatus of claim 25, wherein the thermal controller is configured to control the temperature of at least one of the base member, the sample holder, or the divided biological sample. (Item 27) 26. The apparatus of item 25, wherein the thermal controller comprises a thermal cycler configured to perform a PCR assay. (Item 28) 16. The apparatus according to item 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the apparatus comprises the sample holder. (Item 29) 30. The apparatus of claim 28, wherein the sample holder comprises one or more of a microtiter plate, a substrate comprising a plurality of wells or through-holes, a substrate comprising one or more channels, or a chamber comprising a plurality of beads or spheres containing one or more biological samples. (Item 30) 30. The device of item 28, wherein the plurality of spatially separated reaction regions comprises one or more of a plurality of wells in a substrate, a plurality of through-holes, a plurality of distinct locations on a substrate or within a channel, a plurality of microbeads or microspheres within a reaction volume, and the like. (Item 31) 30. The apparatus of claim 28, wherein at least a portion of the spatially separated reaction areas contain one or more of the biological samples. (Item 32) 32. The apparatus of claim 31, wherein the one or more biological samples 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 nucleic acid sequence or a reference nucleic acid sequence. (Item 33) 30. The apparatus of claim 28, wherein the sample holder comprises a microtiter plate and the reaction area comprises at least 96 wells, at least 384 wells, or at least 1536 wells. (Item 34) 30. The apparatus of claim 28, wherein the sample holder comprises a substrate including a first surface, an opposing second surface, and a plurality of through-holes disposed between the surfaces, the plurality of through-holes configured to contain one or more biological samples. (Item 35) Item 35. The apparatus of item 34, wherein the substrate comprises at least 3096 through-holes or at least 20,000 through-holes. (Item 36) 30. The apparatus of claim 28, wherein the sample holder comprises a series of capillaries configured to pass one or more target molecules or series of molecules. (Item 37) 30. The apparatus of claim 28, wherein the sample holder is configured to perform at least one of a polymerase chain reaction, a sequencing assay, or a capillary electrophoresis assay, a blot assay. (Item 38) 16. The apparatus of claim 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14 or 15, wherein the excitation optical system comprises a sample lens configured to direct the excitation beam towards the base member. (Item 39) Item 39. The apparatus of item 38, wherein the sample lens comprises a field lens configured to extend across a plurality of the spatially separated regions. (Item 40) 40. The apparatus of claim 8, 38 or 39, further comprising a plurality of lenses, each lens being disposed over a respective one of the plurality of reaction areas. (Item 41) 40. The apparatus of claim 8, 38 or 39, wherein the sample lens comprises at least one of a blended lens, a curved mirror, a refractive optical element, or a holographic optical element. (Item 42) 40. The apparatus of claim 8, 38 or 39, wherein the sample lens is configured to provide a telecentric optical system for at least one of the sample holder, the spatially separated reaction regions or the optical sensor. (Item 43) 40. The apparatus of claim 8, 38 or 39, wherein the sample lens comprises a Fresnel lens. (Item 44) 40. The apparatus of claim 8, 38 or 39, wherein the sample lens comprises a plurality of lenses corresponding to a plurality of the reaction regions. (Item 45) 16. The apparatus of claim 7, 8, 11, 12, 13, 14 or 15, wherein the beam splitter is configured to transmit the excitation beam or configured to reflect the excitation beam. (Item 46) 16. The apparatus of claim 7, 8, 11, 12, 13, 14 or 15, wherein the beam splitter comprises a broadband beam splitter characterized by a constant reflectance over a wavelength band of at least 100 nanometers. (Item 47) 16. The apparatus of claim 7, 8, 11, 12, 13, 14 or 15, wherein the beam splitter is characterized by a constant reflectance over the wavelength range of 450 nanometers to 680 nanometers. (Item 48) 16. The apparatus of claim 7, 8, 11, 12, 13, 14 or 15, wherein the beam splitter is characterized by a constant reflectance across the visible light wavelength range of the electromagnetic spectrum. (Item 49) 16. The apparatus of claim 7, 8, 11, 12, 13, 14 or 15, wherein the beam splitter is equipped with a neutral density filter. (Item 50) 16. The apparatus of claim 7, 8, 11, 12, 13, 14 or 15, wherein the beam splitter comprises a 50 / 50 beam splitter. (Item 51) 16. The apparatus of claim 7, 8, 11, 12, 13, 14 or 15, wherein the beam splitter comprises a dichroic beam splitter. (Item 52) 16. The apparatus of claim 7, 8, 11, 12, 13, 14 or 15, wherein the beam splitter comprises a multi-wavelength beam splitter. (Item 53) 16. The apparatus of claim 8, 10, 11, 12, 13, 14 or 15, wherein the first excitation beam and the second excitation beam are temporally split and / or spatially separated. (Item 54) 16. The apparatus of claim 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the first excitation beam and the second excitation beam are produced simultaneously. (Item 55) 16. The apparatus of claim 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the excitation light source comprises one or more of at least one incandescent lamp, at least one gas discharge lamp, at least one light emitting diode, at least one organic light emitting diode, or at least one laser. (Item 56) Item 56. The apparatus of item 55, wherein the at least one gas discharge lamp comprises one or more of a halogen lamp, a xenon lamp, an argon lamp, or a krypton lamp. (Item 57) 56. The apparatus of claim 55, wherein the at least one laser comprises one or more of a diode laser, an argon laser, a xenon laser, an excimer laser, a solid-state laser, a helium-neon laser, or a dye laser. (Item 58) 16. The apparatus of claim 1, wherein the first excitation beam has a first wavelength range such that an intensity, power or energy of the first excitation beam is greater than a first predetermined value, and the second excitation beam has a second wavelength range such that an intensity, power or energy of the second excitation beam is greater than a second predetermined value, the first wavelength being at least one of (1) a central wavelength of the first wavelength range, or (2) a wavelength of maximum electromagnetic field intensity, power or energy of the first wavelength range, and the second wavelength being at least one of (1) a central wavelength of the second wavelength range, or (2) a wavelength of maximum electromagnetic field intensity, power or energy of the second wavelength range. (Item 59) Item 59. The apparatus of item 58, wherein at least one of the central wavelengths is an average wavelength of a corresponding wavelength range. (Item 60) Item 59. The apparatus of item 58, wherein at least one of the predetermined values ​​is less than 20% of a corresponding maximum intensity, power or energy for a corresponding wavelength range. (Item 61) Item 59. The apparatus of item 58, wherein the second predetermined value is equal to the first predetermined value. (Item 62) Item 59. The apparatus of item 58, wherein the first wavelength range does not overlap with the second wavelength range or the first wavelength range only partially overlaps with the second wavelength range. (Item 63) Item 59. The apparatus of item 58, wherein the second wavelength differs from the first wavelength by at least 20 nanometers. (Item 64) Item 59. The apparatus of item 58, wherein at least one of the first wavelength range and the second wavelength range has a value of at least 20 nanometers. (Item 65) 16. The apparatus of item 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the second wavelength differs from the first wavelength by at least 20 nanometers. (Item 66) 16. The apparatus of items 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the excitation source comprises a light source, and the first wavelength and the second wavelength are in the visible light band of the electromagnetic spectrum. (Item 67) 16. The apparatus of items 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the excitation source comprises a light source having a bandwidth of at least 100 nanometers. (Item 68) 16. The apparatus of claim 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the excitation source comprises a plurality of excitation filters movable in and out of the excitation light path. (Item 69) 70. The apparatus of claim 68, wherein at least one of the excitation filters has a wavelength band of at least ±10 nanometers. (Item 70) Item 69. The apparatus of item 68, wherein the excitation filter comprises at least five excitation filters. (Item 71) Item 69. The apparatus of item 68, wherein the excitation filter comprises a plurality of filters that pass a plurality of bands suitable for the fluorescence of one or more of a SYBR® dye or probe, a FAM™ dye or probe, a VIC® dye or probe, a ROX™ dye or probe, or a TAMRA™ dye or probe. (Item 72) 70. The apparatus of claim 68, wherein the excitation filters are mounted on a rotatable filter wheel structure to move each filter in and out of the excitation light path. (Item 73) 16. The apparatus of items 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the excitation source comprises a plurality of individual excitation sources. (Item 74) Item 74. The apparatus of item 73, wherein the plurality of individual excitation sources form a two-dimensional series of individual excitation sources. (Item 75) 16. The apparatus of item 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the optical sensor comprises an array sensor. (Item 76) Item 76. The apparatus of item 75, wherein the array sensor comprises at least one of a complementary metal oxide semiconductor sensor or a charge coupled device sensor. (Item 77) 16. The apparatus of item 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14 or 15, wherein the optical sensor comprises at least two array sensors. (Item 78) 16. The apparatus of claim 2, 3, 4, 6, 11, 12, 13 or 15, further comprising a sensor lens configured to direct luminescence from at least a portion of the biological sample towards the optical sensor. (Item 79) 16. The apparatus of item 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14 or 15, wherein the optical sensor is a complementary metal oxide semiconductor sensor. (Item 80) Item 12. The device of item 2 or 11, wherein the blocking structure is positioned to cooperate with the sensor opening such that the reflected radiation does not impinge on the sensor lens without also being reflected off another surface of the device. (Item 81) 12. The device of claim 2, 8 or 11, further comprising a heated cover having a plurality of openings, the irradiated area comprising an area defined by all of the openings of the heated cover. (Item 82) 16. The apparatus of claim 1, further comprising a heated cover disposed adjacent to the base member and having a plurality of openings configured to correspond to a plurality of the reaction areas. [Brief description of the drawings]

[0003] Embodiments of the invention will be better understood from the following detailed description when read in conjunction with the accompanying drawings, which are merely illustrative and set forth novel and non-obvious aspects of the invention. The drawings include the following figures:

[0004] [Figure 1] FIG. 1 is a schematic diagram of a system according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic diagram of an excitation source according to one embodiment of the present invention. [Diagram 3] FIG. 3 is a normalized spectral plot of various light sources, including a light source according to one embodiment of the present invention. [Figure 4] FIG. 4 is a spectral integration plot over various wavelength ranges of the light source spectrum shown in FIG. [Diagram 5] FIG. 5 is a perspective view of a device housing according to one embodiment of the present invention. [Figure 6] FIG. 6 is a perspective view of a device housing according to one embodiment of the present invention. [Figure 7] FIG. 7 is an illustration of a solid model of an optical sample processing system according to one embodiment of the present invention. [Figure 8] FIG. 8 is an enlarged view of the solid model of the optical system shown in FIG. [Figure 9] FIG. 9 is an exploded view of a portion of the sample processing system shown in FIG. [Figure 10] FIG. 10 is a cross-sectional view of a portion of the optical system shown in FIG. [Figure 11] FIG. 11 is a perspective view of an imaging unit according to one embodiment of the present invention. [Figure 12] FIG. 12 is a cross-sectional view of the imaging unit shown in FIG. [Figure 13] 13 is a bottom perspective view of the imaging unit shown in FIG. [Figure 14] 14 is a bottom perspective view of the imaging unit shown in FIG. [Figure 15] FIG. 15 is an enlarged view of a portion of the imaging unit shown in FIG. [Figure 16] FIG. 16 is an enlarged view of a portion of the imaging unit shown in FIG. [Figure 17] FIG. 17 is an enlarged view of a portion of the imaging unit shown in FIG. [Figure 18] FIG. 18 is a cross-sectional view of the system shown in FIGS. [Figure 19] FIG. 19 is a schematic diagram of a system according to one embodiment of the present invention. [Figure 20] FIG. 20 is a schematic diagram of a system according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] As used herein, the terms "radiation" or "electromagnetic radiation" refer to radiant energy emitted by certain electromagnetic processes, which may include one or more of visible light (e.g., radiant energy characterized by one or more wavelengths between 400 nanometers and 700 nanometers, or between 380 nanometers and 800 nanometers) or invisible electromagnetic radiation (e.g., infrared, near infrared, ultraviolet (UV), x-ray, or gamma ray radiation).

[0006] As used herein, an excitation source refers to an electromagnetic radiation source that may be directed at at least one sample, including one or more chemical compounds, such that the electromagnetic radiation interacts with the at least one sample and produces luminescent electromagnetic radiation indicative of the condition of the at least one sample. The excitation source may include a light source. As used herein, the term "light source" refers to an electromagnetic radiation source that includes an electromagnetic spectrum having a peak or maximum output (e.g., power, energy, or intensity) in the visible light wavelength band of the electromagnetic spectrum (e.g., electromagnetic radiation in the wavelength range of 400 nanometers to 700 nanometers or the wavelength range of 380 nanometers to 800 nanometers). Additionally or alternatively, the excitation source may include electromagnetic radiation in at least a portion of the infrared portion (near infrared, mid-infrared, and / or far infrared) or ultraviolet portion (near ultraviolet and / or extreme ultraviolet) of the electromagnetic spectrum. Additionally or alternatively, the excitation source may include electromagnetic radiation in other wavelength bands of the electromagnetic spectrum (e.g., X-ray and / or radio wave portions of the electromagnetic spectrum). The excitation source may include a single light source, such as, for example, an incandescent lamp, a gas discharge lamp (e.g., a halogen lamp, a xenon lamp, an argon lamp, a krypton lamp, etc.), a light emitting diode (LED), an organic LED (OLED), a laser, etc. The excitation source may include multiple individual light sources (e.g., multiple LEDs or lasers). The excitation source may also include one or more excitation filters, such as a high-pass filter, a low-pass filter, or a band-pass filter. For example, the excitation filter may include a colored filter and / or a dichroic filter. The excitation source includes a single beam or multiple beams that are spatially and / or temporally separated.

[0007] As used herein, "luminescence" refers to electromagnetic radiation produced as a result of the interaction of radiation from an excitation source with one or more samples that contain or are believed to contain one or more chemical and / or biological molecules or compounds of interest. The luminescence may result from reflection, refraction, polarization, absorption and / or other optical effects by the sample on the radiation from the excitation source. For example, the luminescence may include luminescence or fluorescence induced by absorption of the excitation electromagnetic radiation by one or more samples. As used herein, "luminescence" refers to luminescence that includes the electromagnetic spectrum having a peak or maximum power (e.g., power, energy or intensity) in the visible light wavelength band of the electromagnetic spectrum (e.g., electromagnetic radiation in the wavelength range of 420 nanometers to 700 nanometers).

[0008] As used herein, a lens refers to an optical element configured to direct or focus incident electromagnetic radiation to provide a real or virtual image, for example, at a finite distance or at optical infinity, by converging or radially expanding such radiation. A lens may comprise a single optical element having optical power provided by refraction, reflection and / or diffraction of incident electromagnetic radiation. Alternatively, a lens may include a mixed system including multiple optical elements, including, for example, but not limited to, an achromatic lens, a doublet lens, a triplet lens or a camera lens. A lens may be at least partially housed or at least partially enclosed in a lens case or lens mount.

[0009] As used herein, the term "optical power" refers to the ability of a lens or optic to converge or radially expand light and provide a focal point (real or virtual) when placed in air. As used herein, the term "focal length" refers to the inverse of optical power. As used herein, the term "refractive power" or "refractive optical power" refers to the power of a lens or optic or a portion thereof that contributes to refracting incident light in one or more refractive dimensions. Unless otherwise stated, the optical power of a lens, optic, or optical element is from a reference surface associated with the lens or optic (e.g., the primary surface of the optic).

[0010] As used herein, the term "biological sample" refers to a sample or solution containing any type of biochemical element 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, and any sample or solution containing related chemicals or compounds used for the purpose of performing a biological assay, experiment or test. These biochemical elements, 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 biomolecules. A biological sample may contain 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 nucleic acid sequence or a reference nucleic acid sequence. In various embodiments, such biological components may be used in combination with one or more PCR methods and systems for 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 variations.

[0011] According to embodiments of the invention, one or more samples or solutions containing at least one biological target of interest may be contained in, distributed among, or separated among a plurality of small-volume sample or reaction areas (e.g., volumes or areas of 10 nanoliters or less, 1 nanoliter or less, or 100 picoliters or less). Although the reaction areas disclosed herein are generally shown as being contained in wells disposed within a substrate material, reaction areas in other forms of embodiments of the invention may include through-holes or indentations made in the substrate, spots of solution distributed on a substrate surface, reaction areas disposed within test sites or volumes of capillaries or microfluidic systems, or samples or solutions disposed within or on microbeads or microspheres.

[0012] 1, a system, equipment or device 100 for biological analysis includes one or more of an electronic processor, computer or controller 200, a base member, mounting or sample block assembly 300 configured to receive and / or process a biological or biochemical sample, and / or an optical system, equipment or device 400. Without limiting the scope of the invention, the system 100 may include a sequencing device, a polymerase chain reaction (PCR) device (e.g., a real-time PCR (qPCR) device and / or a digital PCR (dPCR) device), a capillary electrophoresis device, a device for providing genotyping information, and the like.

[0013] The electronic processor 200 is configured to control, monitor, and / or accept data from the optical system 400 and / or the base member 300. The electronic processor 200 may be physically integrated with the optical system 400 and / or the base member 300. Additionally or alternatively, the electronic processor 200 may be separate from the optical system 400 and / or the base member 300, such as an external desktop computer, laptop computer, notepad computer, tablet computer, etc. Communication between the electronic processor 200 and the optical system 400 and / or the base member 300 may be achieved directly via a physical connection (e.g., a USB cable, etc.) and / or indirectly via a wireless or network connection (e.g., via a Wi-Fi connection, a local area network, an Internet connection, a cloud connection, etc.). The electronic processor 200 may comprise electronic memory storage including instructions, routines, algorithms, test and / or construction parameters, test and / or experimental data, etc. The electronic processor 200 may be configured, for example, to operate various components of the optical system 400 or to obtain and / or process data provided by the base member 300. For example, the electronic processor 200 may be used to obtain and / or process optical data provided by one or more optical detectors of the optical system 400.

[0014] In certain embodiments, the electronic processor 200 may be integrated into the optical system 400 and / or the base member 300. The electronic processor 200 may communicate with and / or send data to an external computer for further processing using, for example, a hardwire connection, a local area network, an internet connection, a cloud computing system, etc. The external computer may be a physical computer, for example, a desktop computer, a laptop computer, a notepad computer, a tablet computer, etc., and the external computer is located in or near the system 100. Additionally or alternatively, one or both of the external computer and the electronic processor 200 may have a virtual device or system, for example, a cloud computing or storage system. Data may be transferred between the two via a wireless connection, a cloud storage or computing system, etc. Additionally or alternatively, data from the electronic processor 200 (e.g., data from the optical system 400 and / or the base member 300) may be transferred to an external memory storage device, for example, an external hard drive, a USB memory module, a cloud storage system, etc.

[0015] In certain embodiments, the base member 300 is configured to receive a sample holder or sample carrier 305. The sample holder 305 may include a plurality or series of spatially separated reaction areas, sites or locations 308 for containing a corresponding plurality or series of biological or biochemical samples 310. The reaction areas 308 may include any plurality of volumes or locations that isolate or are configured to isolate a plurality of biological or biochemical samples 310. For example, the reaction areas 308 may include a plurality of through-holes or wells in a substrate or assembly (e.g., sample wells in a standard microtiter plate), a plurality of sample beads, microbeads, or microspheres in a channel, capillary, or chamber, a plurality of separate locations in a flow cell, a plurality of sample spots on a substrate surface, or a plurality of wells or openings configured to receive a sample holder (e.g., cavities in a sample block assembly configured to receive a microtiter plate).

[0016] The base member 300 may include a sample block assembly configured to control the temperature of the sample holder 305 and / or the biological sample 310. The sample block assembly 300 may include one or more of a sample block, a Peltier device, or other apparatus for controlling or cycling temperature, and / or a heat sink (e.g., to help stabilize temperature). The base member 300 may include a thermal controller or thermal cycler, for example, for administering or performing a PCR assay.

[0017] The reaction device 300 may include a sample holder 305. At least a portion of the reaction area 308 may include one or more biological samples 310. The biological or biochemical sample 310 may include 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 the target nucleic acid sequence or the reference nucleic acid sequence. The sample holder 305 may be configured to perform at least one of a PCR assay, a sequencing assay, or a capillary electrophoresis assay, a blot assay. In certain embodiments, the sample holder 305 may include one or more of a microtiter plate, a substrate including a plurality of wells or through-holes, a substrate including one or more channels or capillaries, or a chamber including a plurality of beads or spheres containing one or more biological samples. The reaction area 308 may comprise one or more of a plurality of wells in a substrate, a plurality of through-holes, a plurality of distinct locations on a substrate or in a channel or capillary, a plurality of microbeads or microspheres within a reaction volume, etc. The sample holder 305 may comprise a microtiter plate, for example, the reaction area 308 may comprise at least 96 wells, at least 384 wells, or at least 1536 wells.

[0018] In certain embodiments, the sample holder 305 may comprise a substrate including a first surface, an opposing second surface, and a plurality of through-holes disposed between the surfaces, the plurality of through-holes configured to contain one or more biological samples, such as those described in published patent applications US 2014-0242596 and WO 2013 / 138706, which are incorporated by reference herein as if fully set forth herein. In such embodiments, the substrate may include at least 3096 through-holes or at least 20,000 through-holes. In certain embodiments, the sample holder 305 may comprise a series of capillaries configured to pass one or more target molecules or series of molecules.

[0019] In certain embodiments, the system 100 may optionally include a heated or temperature-controlled cover 102, which may be disposed over the sample holder 305 and / or base member 300. For example, the heated cover 102 may be used to prevent condensation on the sample contained in the sample holder 305, which may help maintain optimal light access to the biological sample 310.

[0020] In certain embodiments, the optical system 400 includes an excitation, illumination, radiation, or light source 402 and generates at least a first excitation beam 405a characterized by a first wavelength and a second excitation beam 405b characterized by a second wavelength different from the first wavelength. The optical system 400 further includes an optical sensor or light detector 408 configured to receive luminescence or radiation from one or more biological samples in response to the excitation source 410 and / or in response to one or more of the excitation beams 405a, 405b. The optical system 400 further includes an excitation optical system 410 disposed along an excitation optical path 412 between the excitation source 402 and the one or more biological samples to be illuminated. The optical system 400 further includes an emission optical system 415 disposed along an emission optical path 417 between the samples to be illuminated and the optical sensor 408. In certain embodiments, the optical system 400 may include a beam splitter 420. Optical system 400 may optionally include a beam dump or radiation baffle 422 configured to reduce or prevent reflection of radiation in emission optical path 417 from excitation source 402 that impinges on beam splitter 420 .

[0021] In the embodiment shown in FIG. 1 and other embodiments of the invention disclosed herein, the excitation source 402 comprises a radiation source 425. The radiation source 425 may comprise one or more of at least one incandescent lamp, at least one gas discharge lamp, at least one light emitting diode (LED), at least one organic light emitting diode, and / or at least one laser. For example, the radiation source 425 may include at least one halogen lamp, xenon lamp, argon lamp, krypton lamp, diode laser, argon laser, xenon laser, excimer laser, solid state laser, helium-neon laser, dye laser, or combinations thereof. The radiation source 425 may include a light source characterized by a maximum wavelength or a central wavelength in the visible band of the electromagnetic spectrum. Additionally or alternatively, the radiation source 425 may include an ultraviolet, infrared, or near-infrared source having a corresponding maximum wavelength or central wavelength in one of the wavelength bands of the electromagnetic spectrum. The radiation source 425 may be a broadband source, e.g., a broadband source with a spectral bandwidth of at least 100 nanometers, at least 200 nanometers, or at least 300 nanometers, where the bandwidth is defined as the range over which the intensity, energy, or power output is greater than a predetermined amount (e.g., the predetermined amount is about 1%, 5%, or 10% of the maximum or central wavelength of the radiation source). The excitation source 402 further comprises an excitation source lens 428 configured to condition the emission from the radiation source 425, e.g., to increase the amount of excitation radiation received by the sample holder 305 and / or into the biological sample 310. The excitation source lens 428 may comprise a single lens or may be a mixed lens including two or more elements.

[0022] In certain embodiments, the excitation source 402 further comprises two or more excitation filters 430 movable in and out of the excitation light path 412, for example, for use in combination with the broadband excitation source 402. In such embodiments, different excitation filters 430 may be used to select different wavelength ranges or excitation channels suitable for inducing fluorescence from respective dyes or markers in the biological sample 310. The one or more excitation filters 430 may have a wavelength bandwidth of at least ±10 nanometers or at least ±15 nanometers. The excitation filters 430 may be used to filter out SYBR® dyes or probes, FAM TM Dyes or probes, VIC® dyes or probes, ROX TM Dyes or probes, or TAMRA TM The excitation filter 430 may include multiple filters that pass multiple bands suitable for the fluorescence of one or more of the dyes or probes. The excitation filter 430 may be aligned with a rotatable filter wheel (not shown) or other suitable device or apparatus that provides different excitation channels using the excitation source 402. In certain embodiments, the excitation filter 430 may include at least five filters or at least six filters.

[0023] In certain embodiments, the excitation source 402 may comprise a plurality of individual excitation sources, which may be combined using one or more beam splitters or beam combiners such that radiation from each individual excitation source travels along a common optical path, e.g., along the excitation optical path 412 shown in FIG. 1. Alternatively, at least some of the individual excitation sources may be arranged to provide excitation beams that travel along different non-overlapping optical paths, e.g., to irradiate different reaction regions of the plurality of reaction regions 308. Each individual excitation source may be assigned, activated, or selected, e.g., to irradiate the reaction regions 308 individually, in groups, or all at the same time. In certain embodiments, the individual excitation sources may be arranged in a one-dimensional or two-dimensional array, where one or more of the individual excitation sources are characterized by a maximum or central wavelength that is different from at least one of the other individual excitation sources in the array.

[0024] In certain embodiments, the first excitation beam 405a has a first wavelength range such that the intensity, power or energy of the first excitation beam 405a is greater than a first predetermined value, and the second excitation beam 405b has a second wavelength range such that the intensity, power or energy of the second excitation beam 405b is greater than a second predetermined value. The characteristic wavelength of the excitation beams 405a, 405b may be the central wavelength of the corresponding wavelength range, or may be the wavelength of the maximum electromagnetic field intensity, power or energy of the corresponding wavelength range. The central wavelength of at least one of the excitation beams 405 may be the average wavelength of the corresponding wavelength range. For each excitation beam 405 (e.g., excitation beams 405a, 405b), the aforementioned predetermined value may be less than 20% of the corresponding maximum intensity, power or energy, less than 10% of the corresponding maximum intensity, power or energy, less than 5% of the corresponding maximum intensity, power or energy, or less than 1% of the corresponding maximum intensity, power or energy. The above-mentioned predetermined values ​​may be the same for all excitation beams 405 (e.g., for both excitation beams 405a, 405b), or the above-mentioned predetermined values ​​may be different from each other. In certain embodiments, the wavelength ranges of the first and second excitation beams 405a, 405b do not overlap, while in other embodiments, at least one of the wavelength ranges at least partially overlaps with the other wavelength range. In certain embodiments, the first and second central wavelengths are at least 20 nanometers apart. In certain embodiments, at least one of the first and second wavelength ranges has a value of at least 20 nanometers or at least 30 nanometers.

[0025] The excitation optical system 410 is configured to direct excitation beams 405a, 405b to one or more biological samples. Where applicable, references herein to excitation beams 405a, 405b may apply to embodiments including more than two excitation beams 405. For example, the excitation source 402 may be configured to direct at least five or six excitation beams 405. The excitation beams 405a, 405b may be generated or provided simultaneously, may be temporally split, and / or may be spatially separated (e.g., excitation beam 405a is directed to one reaction area 308 and excitation beam 405b is directed to a different reaction area 308). The excitation beams 405 may be generated sequentially, for example, by sequentially turning on and off individual radiation sources 425 of different colors characterized by different wavelengths, or by sequentially placing different color filters in front of one radiation source 425. Alternatively, the excitation beams 405a, 405b may be generated simultaneously, for example, by using multi-band filters, beam splitters or mirrors, or by combining different individual radiation sources 425, for example, two different color light emitting diodes (LEDs). In some embodiments, the excitation source 402 generates more than two excitation beams 405, and the excitation optical system 410 directs each excitation beam toward one or more biological samples 310.

[0026] 2, the excitation source 402 may have at least five individual radiation sources 425a, 425b, 425c, 425d, 425e that may combine and travel along the excitation light path 412. The excitation source 402 may also include corresponding excitation source lenses 428a, 428b, 428c, 428d, 428e. A number of combiner optical elements 429a, 429b, 429c may be used to combine the radiation from the radiation sources 425a, 425b, 425c, 425d, 425e. The combiner optical elements 429a, 429b, 429c may include one or more of a neutral density filter, a 50 / 50 beam splitter, a dichroic filter or mirror, a cube beam splitter, and the like. It will be understood that the combiner optical element 429a, 429b, 429c is one example of how to combine the individual radiation sources 425, and other combinations and geometries of the individual radiation sources 425 and the combiner optical element 429 are within the scope of embodiments of the present invention. One or more of the individual radiation sources 425a, 425b, 425c, 425d, 425e may be characterized by a different central wavelength and / or wavelength range than the other individual radiation sources 425a, 425b, 425c, 425d, 425e.

[0027] 3-4, the spectral distribution of the radiation source 425 may be selected in a non-obvious manner, such as to allow at least five excitation beams 405 of different colors or excitation channels to be used with one common beam splitter 420 while simultaneously maintaining acceptable or predetermined data across all excitation channels during each cycle of a qPCR assay. As used herein, the term "excitation channel" refers to a number of distinct and respective electromagnetic wavelength bands provided by an excitation source (e.g., excitation source 402) configured to irradiate one or more biological samples (e.g., biological sample 310). As used herein, the term "emission channel" refers to a number of distinct and respective emission wavelength bands through which electromagnetic radiation can pass to an optical sensor or detector (e.g., optical sensor 408).

[0028] FIG. 3 shows the relative energy versus wavelength spectrum for three different radiation sources. The dotted line plot is the spectrum of a halogen lamp (referred to herein as "Radiation Source 1"), characterized by relatively low energy levels in the blue wavelength range of the visible light spectrum, increasing in energy to a peak value at about 670 nanometers. The dash-dotted line spectral plot is a plot of a commercially available LED light source (referred to herein as "Radiation Source 2"), with a peak energy around 450 nanometers, a smaller peak at about 530 nanometers to about 580 nanometers, and then a gradual decrease in energy in the red wavelength range of the visible light spectrum. The solid line plot is the spectrum of another LED light beam (e.g., an exemplary spectrum of excitation source 402) according to an embodiment of the present invention (referred to herein as "Radiation Source 3"). FIG. 4 shows the energy integrated across the various excitation channels for each of the three radiation sources shown in FIG. 3, where the spectra of these channels are typical excitation filters used in the field of qPCR. The wavelength ranges and excitation filter names are shown in Table 1 below, where X1 is excitation channel 1, X2 is excitation channel 2, etc. [Table 1]

[0029] In the field of qPCR, one of the important performance parameters is the total time to obtain emission data for a sample containing multiple target dyes. For example, in some cases, it is desirable to obtain emission data from multiple dyes or probes in one or more emission channels, designated M1 through M6, for each excitation channel used to irradiate the sample (e.g., M1-M6 and X1, M2-M6 and X2, M3-M6 and X3, M4-M6 and X4, M5-M6 and X5, and / or M6 and X6). The inventors have discovered that when radiation source 2 is used in a system having one broadband beam splitter for five or six excitation / emission filter channels (e.g., a combination of excitation channels X1-X6 and emission channels M1-M6), the amount of time to obtain data for excitation channel 5 and / or excitation channel 6 is unacceptably long for certain applications. To improve this situation, it is possible to use one or more narrow band dichroic beam splitters for excitation channels 1 and / or 2 to increase the amount of excitation light received by the sample and the amount of emission received by the sensor (so that in this case the overall optical efficiency is increased by the dichroic beam splitter). However, this precludes the use of a single beam splitter arrangement as shown in FIG. 1, and thus the corresponding advantages of the corresponding single beam splitter structure (e.g. reduced size, cost, complexity) are lost. A better solution has been developed using a light source such as radiation source 3 in combination with a single beam splitter (e.g. a broadband beam splitter such as a 50 / 50 beam splitter, e.g. beam splitter 420). It has been found that the relative energies in excitation channels X1, X5 and / or X6 may be used to identify a suitable excitation source 402 for use with a single beam splitter embodiment to obtain acceptable total integration times for collecting emission data in five or six excitation channels. Using LED radiation source 2 and LED radiation source 3 as examples, the following data shown in Table 2 below may be derived for the data shown in FIGS. [Table 2]

[0030] Based on such data, the inventors have discovered that in certain embodiments, superior performance (e.g., in terms of shorter integration times for channel 1) may be obtained when X1 / X2 is greater than 2.02 (e.g., 3 or greater). Additionally or alternatively, in other embodiments, superior performance (e.g., in terms of shorter integration times for channel 1) may be obtained when X5 / X2 is greater than 0.49 (e.g., 0.9 or greater) and / or when X6 / X2 is greater than 0.38 (e.g., 0.9 or greater). For purposes of the criteria herein, "X1" refers to an excitation channel having a spectral output characterized by a maximum power, energy, or intensity in a wavelength range that includes 455-485 nanometers. "X2" refers to an excitation channel having a spectral output characterized by a maximum power, energy, or intensity in a wavelength range that includes 510-530 nanometers. "X5" refers to an excitation channel having a spectral output characterized by a maximum power, energy, or intensity in a wavelength range that includes 630.5-649.5 nanometers. "X6" means an excitation channel having a spectral output characterized by a maximum power, energy or intensity in the wavelength band between 650 and 674 nanometers inclusive.

[0031] Referring again to FIG. 1, during operation toward the base member 300 for processing a sample, for example, if a sample holder 305 is present, the excitation beam 405 is directed along the excitation optical path 412 toward the reaction region 308. If present, the radiation source lens 428 is configured to condition the excitation beam 405, for example, to capture and direct a majority of the radiation emitted from the excitation source 402. In certain embodiments, one or more mirrors 432 (e.g., folded mirrors) may be incorporated along the excitation optical path 412, for example, to further compact the optical system 400 and / or provide a given package size. Although FIG. 1 shows one mirror 432, additional mirrors may be used, for example, to meet package design constraints. Additional lenses may be positioned near the sample holder 305, for example, to further condition the excitation beam 405 and / or the corresponding emission light from the biological sample contained in the one or more reaction regions, as described in more detail herein below.

[0032] The emission optical system 415 is configured to direct emission light from one or more biological samples to the optical sensor 408. At least a portion of the emission light may include fluorescent emission light from at least some of the biological samples in response to the at least one excitation beam 405. Additionally or alternatively, at least a portion of the emission light includes radiation from the at least one excitation beam 405 that is reflected, refracted, diffracted, scattered, or polarized by at least some of the biological samples. In certain embodiments, the emission optical system 415 includes one or more emission filters 435 configured to block excitation radiation that is reflected or scattered, for example, into the emission optical path 417. In certain embodiments, for each excitation filter 430, there is a corresponding emission filter 435. Referring to FIG. 8, in certain embodiments, the excitation filters 430 are arranged in an excitation filter wheel 431 and / or the emission filters 435 are arranged in an emission filter wheel 436.

[0033] In certain embodiments, the luminescence optical system 415 includes a sensor lens 438 configured to direct luminescence from at least a portion of the biological sample to the optical sensor 408. The optical sensor 408 may include a single sensor element, such as a photodiode detector or a photomultiplier tube. Additionally or alternatively, the optical sensor 408 may include an array sensor including a series of sensors or pixels. The array sensor 408 may include one or more of a complementary metal oxide semiconductor sensor (CMOS), a charge-coupled device (CCD) sensor, a plurality of photodiode detectors, a plurality of photomultiplier tubes, and the like. The sensor lens 438 may be configured to generate an image from luminescence from one or more of the plurality of biological samples 310. In certain embodiments, the optical sensor 408 includes two or more array sensors 408, such as two or more images are generated from luminescence from one or more of the plurality of biological samples 310. In such an embodiment, the luminescence from one or more of the plurality of biological samples 310 may be split to obtain two signals for one or more of the plurality of biological samples 310. In certain embodiments, the optical sensor includes at least two array sensors.

[0034] The beam splitter 420 is disposed along both the excitation light path 412 and the emission light path 417 and is configured to accept both the first and second excitation beams 405a, 405b during operation. In the illustrated embodiment shown in FIG. 1, the beam splitter 420 is configured to transmit the excitation beam 405 and reflect the emission from the biological sample 310. Alternatively, the beam splitter 420 may be configured to reflect the excitation beam and transmit the emission from the biological sample 310. In certain embodiments, the beam splitter 420 comprises a broadband beam splitter having the same or nearly the same reflectivity for all or most of the excitation beam 405 (e.g., in the illustrated embodiment, excitation beams 405a, 405b) provided by the excitation source 402 and directed to the reaction region 308. For example, beam splitter 420 may be a broadband beam splitter characterized by a constant or nearly constant reflectance over at least a 100 nanometer wavelength band, at least a 200 nanometer wavelength band, or the visible light wavelength band, the visible and near-IR wavelength bands of the electromagnetic spectrum, or the wavelength band from 450 nanometers to 680 nanometers. In certain embodiments, beam splitter 420 is a neutral density filter, e.g., a filter having about 20%, 50%, or 80% reflectance over the visible light wavelength band of the electromagnetic spectrum. In certain embodiments, beam splitter 420 is a dichroic beam splitter that is transmissive or reflective over one or more selected wavelength ranges, e.g., a multi-band beam splitter that is transmissive and / or reflective over more than one wavelength band centered at or near the peak wavelength of excitation beam 405.

[0035] In certain embodiments, the beam splitter 420 is a beam splitter structure that accepts some or all of the multiple excitation beams 405 (e.g., excitation beams 405a, 405b), either alone or in combination with a beam dump 422. Each excitation beam may be referred to as an excitation channel and may be used alone or in combination to excite a different fluorescent dye or probe molecule in one or more biological samples 310. In contrast, many conventional systems and devices, for example in the field of qPCR, provide multiple excitation beams by using separate beam splitters and / or beam dumps for each excitation channel and / or each emission channel of the system or device. In such conventional systems and devices, color-selective dichroic filters are typically used in at least some of the excitation channels to increase the amount of radiation received by the sample. Disadvantages of systems and devices that use different beam splitters and / or beam dumps for each channel include increased size, cost, complexity, and response time (e.g., due to the increased mass that must be moved or rotated when changing between excitation and / or emission channels). The inventors have discovered that these multiple beam splitters and / or beam dumps can be replaced with a single beam splitter 420 and / or a single beam dump 422 while maintaining acceptable performance for a given system or apparatus, for example, by appropriately selecting the spectral distribution of the excitation source 402 and / or by configuring the system or apparatus to reduce the amount of stray light or undesired radiation received by the optical sensor 408 (as described further herein). Thus, embodiments of the present invention may be used to provide systems and apparatus that have reduced size, cost, complexity and response times compared to conventional systems and apparatus.

[0036] 5 and 6, in certain embodiments, system 100 comprises an apparatus housing 105 and a sample holder drawer 110, which includes a base member 300 and is configured to receive, hold or contain a sample holder 305 during use and position the sample holder 305 in a position to provide optical connection with an optical system 400. With drawer 110 in a closed position (FIG. 6), housing 105 may be configured to contain or encase sample processing system 300 and optical system 400. In certain embodiments, housing 105 may be configured to contain or encase all or a portion of electronic processor 200.

[0037] 7-9, in certain embodiments, optical system 400 may further include lens 440 and / or lens array 442, which may include a plurality of lenses corresponding to each reaction area 308 of sample holder 305. Lens 440 may include a field lens, which may be configured to provide a telecentric optical system for at least one of sample holder 305, reaction area 308, lens array 442, or optical sensor 408. As shown in the embodiments illustrated in FIGS. 7 and 9, lens 440 may include a Fresnel lens.

[0038] 7 and 9, in certain embodiments, the base member 300 includes a sample block assembly 300 including a sample block 302, a temperature controller 303 (e.g., a Peltier device 303), and a heat sink 304. The sample block assembly 300 may be configured to provide thermal control or cycling (e.g., to provide a PCR assay or temperature profile), maintain the temperature of the sample holder 305 or biological sample 310, and / or otherwise maintain, control, regulate, or cycle the heat flow or temperature of the sample holder 305 or biological sample 310.

[0039] 10-14, in certain embodiments, the optical system 400 includes an imaging unit 445 that includes an optical sensor circuit board 448, a sensor lens 438 (which may be a mixed lens, as shown in FIG. 10), an inner lens mount 449, an outer lens mount 450, a threaded housing 452, and a focus gear 455. The optical sensor circuit board 448, the threaded housing 452, and the sensor lens 438 may together create a cavity 458 that surrounds or receives the optical sensor 408 and may be configured to block external light impinging on the optical sensor 408 from entering the sensor lens 438. The outer lens mount 450 has an outer surface that includes gear teeth 460 that may be movably or slidably engaged with the teeth of the focus gear 455 via a resilient element (not shown), e.g., a spring. In certain embodiments, the focus gear 455 moves or slides along a slot 462 in a plate 465, as shown in Figure 14. The inner lens mount 449 has a threaded portion 468 that engages or mates with a threaded portion of the threaded housing 452.

[0040] The inner lens mount 449 may be fixedly attached to the outer lens mount 450, while the threaded housing 452 is fixedly attached to the optical sensor circuit board 448. The inner lens mount 449 may be movably or rotatably attached to the threaded housing 452. Thus, the focus gear 455 and the outer lens mount 450 may engage such that as the focus gear 455 rotates, the outer lens mount 450 also rotates. This then causes the inner lens mount 449 and the sensor lens 438 to move along the optical axis of the sensor lens 438 via the inner lens mount 449 and the threaded portion of the threaded housing 452. In this manner, the focus of the sensor lens 438 may be adjusted without directly engaging the sensor lens 438 or its associated mounts 449, 450 embedded in the very compact optical system 400. The engagement with the focus gear 455 may be manual or automatic, for example, using a motor (not shown), for example, a stepper motor or a DC motor.

[0041] 11 and 13-17, in certain embodiments, the imaging unit 445 further includes a locking device or mechanism 470. The locking device 470 includes an edge or tooth 472 that may slidably engage between two teeth of the focus gear 455 (see FIGS. 15-17). As shown in FIGS. 15 and 16, the locking device 470 may have a first position (FIG. 15) in which the focus gear 455 is free to rotate and adjust the focus of the sensor lens 438, and a second position (FIG. 14) in which the focus gear 455 is locked in place and prevented or inhibited from rotating. In this manner, the focus of the sensor lens 438 may be advantageously locked while avoiding the threaded portion from moving with the threaded portion 468 of the inner lens mount 449, which may be damaged, and the sensor lens 438 may be prevented from being subsequently refocused after being locked in place. Operation of the locking device 470 may be manual or in an automated manner. In certain embodiments, the locking mechanism 470 further includes a resilient element, such as a spring (not shown), and rotation of the focus gear 455 may be accomplished by exceeding a threshold force created by the resilient element.

[0042] 18, the optical system 400 may further include an optical housing 477. In a particular embodiment, the optical system 400 includes a radiation shield 475 having a sensor opening 478 arranged along the emission light path 417, and at least one blocking structure 480 arranged to cooperate with the sensor opening 478, such that the only radiation reflected from the excitation beam 405 at the irradiated region 482 and passing through the sensor opening 478 is the radiation that is also reflected from at least one other surface or inside the optical housing 477. In other words, the radiation shield 475 is configured to block radiation reflected from the excitation beam 405 at the irradiated region 482 from passing directly through the opening 478, and thus preventing it from passing through the sensor lens 438 and toward the optical detection unit 408. In a particular embodiment, the irradiated region 482 is defined by all the openings 483 of the heated cover 102 corresponding to the plurality of reaction regions 308.

[0043] In the depicted embodiment of FIG. 18 , the blocking structure 480 comprises a ledge 480. Dotted or dashed light rays 484a and 484b may be used to illustrate the effectiveness of the blocking structure 480 in preventing light reflected directly from the illuminated area 482 from passing through the sensor opening 478 and toward the sensor lens 438 and / or optical sensor 408. Light ray 484a starts at the edge of the illuminated area 482 and just passes through the ledge 480, but does not pass through the sensor opening 478. Light ray 484b is another light ray that starts at the same edge of the illuminated area 482 and is blocked by the ledge 480. As can be seen, the light rays that may enter through the sensor opening 478 are not due to the presence of the ledge 480.

[0044] 18, in certain embodiments, the optical system 400 may further comprise an energy or force detection unit, which includes a force or energy sensor 490 optically coupled to one end of a light pipe 492. An opposing end 493 of the light pipe 492 is configured to be illuminated by an excitation beam 405. The light pipe end 493 may be illuminated directly by radiation contained in the excitation beam 405 or indirectly by radiation scattered by, for example, a diffusing surface. In certain embodiments, the sensor 490 is positioned outside the excitation light path 412 from the excitation source 402. Additionally or alternatively, the sensor 490 may be positioned outside the optical housing 477 and / or at a remote location outside the device housing 105. In the illustrated embodiment shown in FIG. 18, the light pipe end 493 may be positioned near or adjacent to the mirror 432 and oriented such that the surface of the light pipe is perpendicular or nearly perpendicular to the surface of the mirror 432 that reflects the excitation beam 405. The inventors have discovered that when oriented in this manner, only a small amount of energy or force is intercepted by light pipe 492 for purposes of monitoring the energy or force of excitation beam 405. Advantageously, by locating sensor 490 outside the optical path of the excitation beam, a more compact optical system 400 may be provided.

[0045] In certain embodiments, the light pipe 492 comprises a fiber or a bundle of fibers. Additionally or alternatively, the light pipe 492 may comprise a rod made from a transparent or transmissive material such as glass, plexiglass, or a polymeric material such as acrylic.

[0046] 19 and 20, in certain embodiments, the device 100 includes a position light source 500 configured to emit radiation 502 and a corresponding position sensor 505 configured to receive the radiation 502 from the position light source 500. The position light source 500 and the position sensor 505 may be configured to generate a position signal indicative of a position of an optical element 435 disposed along an optical path. In certain embodiments, the device 100 may further include a radiation shield 510 configured to at least partially block the radiation 502 from the position light source 500.

[0047] The above presents a full, clear, precise, and accurate description of the best mode contemplated for carrying out this invention, and of the manner and mode of making and using it, in such a manner as to enable any person skilled in the art to make and use the invention. However, the invention permits modifications and alternative constructions which are fully equivalent from the above. Consequently, it is not intended that the invention be limited to the particular embodiments disclosed. On the contrary, it is intended to cover modifications and alternative constructions which are within the spirit and scope of the invention as generally expressed by the following claims which specifically point out and separately claim certain aspects of the invention.

[0048] Exemplary systems for the methods related to various embodiments described in this document include those described in the following applications: · U.S. Design Patent Application No. 29 / 516,847, filed February 6, 2015; · U.S. Design Patent Application No. 29 / 516,883, filed February 6, 2015; · U.S. Provisional Patent Application No. 62 / 112,910, filed February 6, 2015; · U.S. Provisional Patent Application No. 62 / 113,006, filed February 6, 2015; · U.S. Provisional Patent Application No. 62 / 113,183, filed February 6, 2015; · U.S. Provisional Patent Application No. 62 / 113,077, filed February 6, 2015; · U.S. Provisional Patent Application No. 62 / 113,058, filed February 6, 2015; · U.S. Provisional Patent Application No. 62 / 112,964, filed February 6, 2015; · U.S. Provisional Patent Application No. 62 / 113,118, filed February 6, 2015; · U.S. Provisional Patent Application No. 62 / 113,212, filed February 6, 2015; U.S. Patent Application No. __, filed February 5, 2016 (Life Technologies Docket No. LT01023); U.S. Patent Application No. __, filed February 5, 2016 (Life Technologies Docket No. LT01024); U.S. Patent Application No. __, filed February 5, 2016 (Life Technologies Docket No. LT01025); U.S. Patent Application No. __, filed February 5, 2016 (Life Technologies Docket No. LT01028); U.S. Patent Application No. __, filed February 5, 2016 (Life Technologies Docket No. LT01029); U.S. Patent Application No. __, filed February 5, 2016 (Life Technologies Docket No. LT01032); · U.S. Patent Application No. __, filed February 5, 2016 (Life Technologies Docket No. LT01033). all of which are also incorporated herein by reference in their entireties.

Claims

1. 1. An apparatus for biological analysis, the apparatus comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an excitation source configured to emit radiation and to generate an excitation beam; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path between the excitation source and the sample holder; an optical system disposed along an optical path between the base member and the optical sensor; a sensor lens configured to direct luminescence from at least a portion of the biological sample toward the optical sensor; a temperature-controlled cover having an irradiated surface disposed across the excitation light path, the irradiated surface configured to generate reflected radiation including the radiation from the excitation source reflected by the irradiated surface; A radiation shield, the radiation shield comprising: a sensor opening disposed across the emitted light path; Blocking structure and A radiation shield comprising: Equipped with the illuminated surface and the sensor opening and the blocking structure are positioned such that the reflected radiation from the illuminated surface that does not reflect off another surface of the device does not pass through the sensor opening and the blocking structure and is not received by the optical sensor; the illuminated surface is illuminated by the radiation from the excitation source; At least a portion of the emitted light comprises radiation from at least one of the excitation beam that is reflected, refracted, diffracted, scattered, or polarized by at least a portion of the biological sample.

2. 1. An apparatus for biological analysis, the apparatus comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an excitation source configured to emit radiation; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path between the excitation source and the sample holder; an optical system disposed along an optical path between the base member and the optical sensor; a sensor lens configured to direct luminescence from at least a portion of the biological sample toward the optical sensor; a temperature-controlled cover having an irradiated surface disposed across the excitation light path, the irradiated surface configured to generate reflected radiation including the radiation from the excitation source reflected by the irradiated surface; A radiation shield, the radiation shield comprising: a sensor opening disposed across the emitted light path; Blocking structure and a radiation shield comprising: a mirror disposed along the excitation light path between the base member and a beam splitter; Equipped with the illuminated surface and the sensor opening and the blocking structure are positioned such that the reflected radiation from the illuminated surface that does not reflect off another surface of the device does not pass through the sensor opening and the blocking structure and is not received by the optical sensor; The illuminated surface is illuminated by the radiation from the excitation source.

3. 1. An apparatus for biological analysis, the apparatus comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an excitation source configured to emit radiation; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path between the excitation source and the sample holder; an optical system disposed along an optical path between the base member and the optical sensor; a sensor lens configured to direct luminescence from at least a portion of the biological sample toward the optical sensor; a temperature-controlled cover having an irradiated surface disposed across the excitation light path, the irradiated surface configured to generate reflected radiation including the radiation from the excitation source reflected by the irradiated surface; A radiation shield, the radiation shield comprising: a sensor opening disposed across the emitted light path; Blocking structure and A radiation shield comprising: Equipped with the illuminated surface and the sensor opening and the blocking structure are positioned such that the reflected radiation from the illuminated surface that does not reflect off another surface of the device does not pass through the sensor opening and the blocking structure and is not received by the optical sensor; the illuminated surface is illuminated by the radiation from the excitation source; The apparatus, wherein the base member comprises a thermal controller.

4. The apparatus of claim 3 , wherein the thermal controller is configured to control a temperature of at least one of the base member, the sample holder, or the aliquoted biological sample.

5. The apparatus of claim 3 , wherein the thermal controller comprises a thermal cycler configured to perform a PCR assay.

6. 1. An apparatus for biological analysis, the apparatus comprising: a base member configured to receive a sample holder including a plurality of spatially separated reaction areas for processing one or more biological samples; an excitation source configured to emit radiation; an optical sensor configured to receive luminescence from the biological sample in response to the excitation source; an excitation optical system disposed along an excitation optical path between the excitation source and the sample holder; an optical system disposed along an optical path between the base member and the optical sensor; a sensor lens configured to direct luminescence from at least a portion of the biological sample toward the optical sensor; a temperature-controlled cover having an irradiated surface disposed across the excitation light path, the irradiated surface configured to generate reflected radiation including the radiation from the excitation source reflected by the irradiated surface; A radiation shield, the radiation shield comprising: a sensor opening disposed across the emitted light path; Blocking structure and A radiation shield comprising: Equipped with the illuminated surface and the sensor opening and the blocking structure are positioned such that the reflected radiation from the illuminated surface that does not reflect off another surface of the device does not pass through the sensor opening and the blocking structure, and such that the reflected radiation from the illuminated surface that does not reflect off another surface of the device is not received by the optical sensor; the illuminated surface is illuminated by the radiation from the excitation source; The apparatus includes the sample holder.

7. 7. The apparatus of claim 6, wherein the sample holder comprises one or more of a microtiter plate, a substrate comprising multiple wells or multiple through-holes, a substrate comprising one or more channels, or a chamber containing multiple beads or spheres that contain the one or more biological samples.

8. 7. The device of claim 6, wherein the multiple spatially separated reaction regions comprise one or more of multiple wells or multiple through-holes in a substrate, multiple distinct locations on a substrate or in a channel, or multiple beads or spheres within a reaction volume.

9. The device of claim 6 , wherein at least a portion of the plurality of spatially separated reaction regions contain the one or more biological samples.

10. 10. The apparatus of claim 9, wherein the one or more biological samples 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 nucleic acid sequence or a reference nucleic acid sequence.

11. 7. The apparatus of claim 6, wherein the sample holder comprises a microtiter plate and the plurality of reaction regions comprises at least 96 wells, at least 384 wells, or at least 1536 wells.

12. 7. The apparatus of claim 6, wherein the sample holder comprises a substrate including a first surface, an opposing second surface, and a plurality of through-holes disposed between the first surface and the second surface, the plurality of through-holes configured to contain the one or more biological samples.

13. 13. The apparatus of claim 12, wherein the substrate comprises at least 3096 through-holes or at least 20,000 through-holes.

14. The apparatus of claim 6 , wherein the sample holder comprises an array of capillaries configured to pass one or more target molecules or sets of molecules.

15. 7. The apparatus of claim 6, wherein the sample holder is configured to perform at least one of a polymerase chain reaction, a sequencing assay, a capillary electrophoresis assay, or a blot assay.

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