Device, system and method for detecting fluorescence

Abstract

The present disclosure provides devices, systems, and methods for detecting presence of a plurality of fluorescent signals in a detection well for sample analysis. The systems can include low-cost light sources such as light emitting diodes (LEDs). The systems can include light filters and normalization photodetector that are used in conjunction with the LEDs. The systems can include a one or more fluorescence detectors that can detect fluorescent signals from the detection well. The methods disclosed herein comprise directing to a detection well different groups of excitation light having a plurality of excitation wavelengths, wherein the directing different groups of the excitation light is temporally separated. The method further comprises detecting in different groups a plurality of emission lights generated in the detection well, the emission lights having a plurality of emission wavelengths, wherein the detection of the emission light from different groups is temporally separated.

Description

[0001] DEVICE, SYSTEM AND METHOD FOR DETECTING FLUORESCENCE CROSS REFERENCE TO RELATED APPLICATIONS

[0002] Pursuant to 35 U. S. C. § 119(e), this application claims priority to the filing date of the United States Provisional Patent Application Serial No. 63 / 756,757, filed February 10, 2025, and the United States Provisional Patent Application Serial No. 63 / 853,270, filed July 29, 2025, the disclosures of which applications are herein incorporated by reference in their entireties.

[0003] INTRODUCTION

[0004] A fluorometer or a fluorescence detector is a device that measures the fluorescence which occurs when light of specific wavelength excites electrons in a sample, and the electrons in that sample instantly emit light of a longer wavelength. A common fluorometer has a light source for exciting the electrons. The light emitted from the light source is separated into a light beam of a specific excitation wavelength, and then passed through a sample. The sample may contain a molecule which absorbs the specific excitation wavelength and transitions to an excited state and emits fluorescence upon return to a relaxed state. The emission wavelength emitted from the molecule is converted into an electrical signal by a photodetector. The electrical signal is proportional to the intensity of the emission wavelength.

[0005] Throughout the years there has been development in fluorescence detecting devices. U. S. Pat. No. 6,563,581 provides a system for detecting fluorescence emitted from a plurality of samples in a sample tray. Existing systems are typically not very cost-effective or efficient. Therefore, there is a need for an efficient device and method for fluorescence detection.

[0006] SUMMARY

[0007] The present disclosure provides devices, systems, and methods for detecting presence or absence of fluorescent signals in a plurality of detection wells in a cartridge. The devices, systems, and methods of the present disclosure, for detecting presence or absence of fluorescent signals may involve multiple light sources emitting excitation lights at multiple excitation wavelengths to generate excitation light for fluorophores.

[0008] In particular embodiments, which may combine the features of some or all of the above embodiments, a method is disclosed for detecting presence of a plurality of fluorescent signals in a detection well, the method including: generating, by one or more light sources, excitation light including one or more wavelengths or bands of excitation wavelengths, wherein the one or more wavelengths or bands of excitation wavelengths are distributed into a plurality of groups such thateach group is associated with one or more of the wavelengths or bands of excitation wavelengths; providing the excitation light to the detection well by sequentially illuminating the detection well over sub-intervals within an interrogation time interval, wherein each sub-interval is correlated with a respective group of the plurality of groups such that the illumination provided to the detection well during each sub-interval includes the one or more wavelengths or bands of excitation wavelengths associated with the respective group; generating, by a sample disposed in the detection well based on illuminating the detection well, emission light over the interrogation time interval; and detecting, by one or more photodetectors, the emission light as the plurality of fluorescent signals over the interrogation time interval.

[0009] In particular embodiments, which may combine the features of some or all of the above embodiments, the emission light includes one or more wavelengths or bands of emission wavelengths respectively correlated with the one or more wavelengths or bands of excitation wavelengths provided to the detection well, and wherein the emission light is generated by the sample over the interrogation time interval, the generation further includes sequentially emitting, during each sub-interval, the one or more wavelengths or bands of emission wavelengths correlated with the respective one or more wavelengths or bands of excitation wavelengths and associated with the respective group. In particular embodiments, which may combine the features of some or all of the above embodiments, detecting the emission light further includes: separating the one or more wavelengths or bands of emission wavelengths generated by the sample by selectively filtering the emission light based on the plurality of groups; and directing the one or more wavelengths or bands of emission wavelengths of the emission light associated with each group to a separate photodetector of the one or more photodetectors for detection, wherein each wavelength or band of emission wavelengths is detected as a corresponding fluorescent signal of the plurality of fluorescent signals. In particular embodiments, which may combine the features of some or all of the above embodiments, the method includes: amplifying, by one or more amplifiers, the plurality of fluorescent signals detected by the one or more photodetectors; multiplexing, by a multiplexer, the plurality of amplified fluorescent signals; and converting, by an analog-to-digital converter, the multiplexed plurality of amplified fluorescent signals into a digital stream. In particular embodiments, which may combine the features of some or all of the above embodiments, the method includes: normalizing or adjusting, by a normalization photodetector, an intensity of the excitation light generated by the one or more light sources based on sampling at least a portion of the excitation light.In particular embodiments, which may combine the features of some or all of the above embodiments, at least one group of the plurality of groups includes a plurality of wavelengths or a plurality of bands of excitation wavelengths. In particular embodiments, which may combine the features of some or all of the above embodiments, at least two of the plurality of wavelengths or plurality of bands of excitation wavelengths associated with the at least one group are simultaneously provided to the detection well during the sub-interval associated with the at least one group. In particular embodiments, which may combine the features of some or all of the above embodiments, the one or more light sources include "n" light sources configured to generate "n" wavelengths or “n” bands of excitation wavelengths.

[0010] In particular embodiments, which may combine the features of some or all of the above embodiments, the "n" light sources are at least partially integrated into one or more light source components. In particular embodiments, which may combine the features of some or all of the above embodiments, the plurality of groups includes "g" groups so that the “n” wavelengths or "n" bands of excitation wavelengths are distributed into the "g" groups. In particular embodiments, which may combine the features of some or all of the above embodiments, generating the excitation light further includes sequentially activating the "g" groups of the "n" light sources over "g" sub-intervals within the interrogation time interval. In particular embodiments, which may combine the features of some or all of the above embodiments, a g:n correlation between the number of groups and the number of light sources is 1:1, 1:2, 1:3, 1:4, 2:3, 2:5, 2:7, 3:4, 3:5, 3:7, or 3:8.

[0011] In particular embodiments, which may combine the features of some or all of the above embodiments, generating the excitation light further includes, during a sub-interval within the interrogation time interval, simultaneously activating each light source associated with a group corresponding to the sub-interval. In particular embodiments, which may combine the features of some or all of the above embodiments, each band of excitation wavelengths includes one or more excitation wavelengths, and wherein each band of emission wavelengths includes one or more emission wavelengths.

[0012] In some cases, each group of excitation light contains light of one wavelength, which may be the same or different in different groups, and emission light corresponding to different groups of excitation light includes two or more emission wavelengths. Thus, in some cases, in one group of excitation light, a light of a single wavelength produces emission light comprising light of two or more wavelengths. This can be achieved by using different fluorescent dyes that have the same or similar excitation wavelength but different emission wavelengths.In particular embodiments, which may combine the features of some or all of the above embodiments, the sub-intervals within the interrogation time interval are non-overlapping in time. In particular embodiments, which may combine the features of some or all of the above embodiments, the distribution of the bands of excitation wavelengths into the plurality of groups is based on one or more of: (a) increasing a spectral separation of the bands of excitation wavelengths; (b) increasing a spectral separation of the bands of emission wavelengths; or (c) increasing a signal-to-noise ratio of detection. In particular embodiments, which may combine the features of some or all of the above embodiments, eight wavelengths or eight bands of excitation wavelengths are distributed into three groups. In particular embodiments, which may combine the features of some or all of the above embodiments, a first group of the three groups includes a first, a fourth, and a sixth excitation wavelength or band of excitation wavelengths; wherein a second group of the three groups includes a second, a fifth, and an eighth wavelength or band of excitation wavelengths; and wherein a third group of the three groups includes a third and a seventh wavelength or band of excitation wavelengths. In particular embodiments, which may combine the features of some or all of the above embodiments, the wavelengths or bands of excitation wavelengths are numbered based on arranging the eight wavelengths or bands of excitation wavelengths in order of increasing wavelength.

[0013] In particular embodiments, which may combine the features of some or all of the above embodiments, one or more steps of the method associated with detecting the presence of a plurality of fluorescence signals in a detection well are applied to a plurality of detection wells.

[0014] In particular embodiments, which may combine the features of some or all of the above embodiments, a system is disclosed for detecting presence of a plurality of fluorescent signals in a detection well, the system including: one or more light sources configured to generate excitation light including one or more wavelengths or bands of excitation wavelengths, wherein the one or more wavelengths or bands of excitation wavelengths are distributed into a plurality of groups such that each group is associated with one or more wavelengths or one or more of the bands of excitation wavelengths; a first conduit for transmitting the excitation light to the detection well, wherein the one or more light sources are configured to sequentially illuminate the detection well over sub-intervals within an interrogation time interval, wherein each sub-interval is correlated with a respective group of the plurality of groups such that the illumination transmitted to the detection well during each sub-interval includes the one or more wavelengths or bands of excitation wavelengths associated with the respective group; one or more photodetectors; and asecond conduit for transmitting, to the one or more photodetectors, emission light generated by a sample disposed in the detection well over the interrogation time interval.

[0015] In particular embodiments, which may combine the features of some or all of the above embodiments, the emission light includes one or more wavelengths or bands of emission wavelengths respectively correlated with the one or more wavelengths or bands of excitation wavelengths provided to the detection well, and wherein the emission light is generated by the sample over the interrogation time interval, the generation further includes sequentially emitting, during each sub-interval, the one or more wavelengths or bands of emission wavelengths correlated with the respective one or more wavelengths or bands of excitation wavelengths and associated with the respective group. In particular embodiments, which may combine the features of some or all of the above embodiments, a waveguide is configured as the first conduit to transmit the excitation light to the detection well. In particular embodiments, which may combine the features of some or all of the above embodiments, the waveguide is further configured as the second conduit to transmit the emission light away from the detection well. In particular embodiments, which may combine the features of some or all of the above embodiments, the system includes one or more filters configured to separate the one or more wavelengths or bands of emission wavelengths generated by the sample by selectively filtering the emission light based on the plurality of groups.

[0016] In particular embodiments, which may combine the features of some or all of the above embodiments, the system includes a plurality of photodetectors, each photodetector associated with a respective group of the plurality of groups, wherein the system is configured to direct the bands of emission wavelengths associated with each group to a corresponding photodetector of the one or more photodetectors for detection, and wherein each band of emission wavelengths is detected as a corresponding fluorescent signal of the plurality of fluorescent signals.

[0017] In particular embodiments, which may combine the features of some or all of the above embodiments, the system includes a plurality of photodetectors, each photodetector configured to detect an emission light having a wavelength or a band of wavelength that is produced from an excitation wavelength or a band of excitation wavelength with a group of excitation light such that excitation light of two or more wavelengths or bands of wavelengths produced by excitation of light from one group are detected by different photodetectors. Thus, the system is configured to direct one wavelength or a band bands of emission wavelengths associated with each group to a photodetector of the one or more photodetectors for detection, and wherein each band of emissionwavelengths is detected as a corresponding fluorescent signal of the plurality of fluorescent signals.

[0018] In particular embodiments, which may combine the features of some or all of the above embodiments, the system includes one or more amplifiers configured to amplify the plurality of fluorescent signals detected by the one or more photodetectors. In particular embodiments, which may combine the features of some or all of the above embodiments, the system includes a multiplexer configured to multiplex the plurality of amplified fluorescent signals. In particular embodiments, which may combine the features of some or all of the above embodiments, the system includes an analog-to-digital converter configured to convert the multiplexed plurality of amplified fluorescent signals into a digital stream. In particular embodiments, which may combine the features of some or all of the above embodiments, the system includes a normalization photodetector configured to normalize or adjust an intensity of the excitation light generated by the one or more light sources based on sampling at least a portion of the excitation light.

[0019] In particular embodiments, which may combine the features of some or all of the above embodiments, at least one group of the plurality of groups includes a plurality of wavelengths or a plurality of bands of excitation wavelengths. In particular embodiments, which may combine the features of some or all of the above embodiments, at least two of the plurality of wavelengths or bands of excitation wavelengths associated with the at least one group are simultaneously provided to the detection well during the sub-interval associated with the at least one group. In particular embodiments, which may combine the features of some or all of the above embodiments, the one or more light sources include "n" light sources configured to generate "n" wavelengths or “n” bands of excitation wavelengths.

[0020] In particular embodiments, which may combine the features of some or all of the above embodiments, the "n" light sources are at least partially integrated into one or more light source components. In particular embodiments, which may combine the features of some or all of the above embodiments, the plurality of groups includes "g" groups so that the "n" wavelengths or “n” bands of excitation wavelengths are distributed into the "g" groups. In particular embodiments, which may combine the features of some or all of the above embodiments, generating the excitation light further includes sequentially activating the "g" groups of the "n" light sources over "g" sub-intervals within the interrogation time interval. In particular embodiments, which may combine the features of some or all of the above embodiments, a g:n correlation between the number of groups and the number of light sources is 1:1, 1:2, 1:3, 1:4, 2:3, 2:5, 2:7, 3:4, 3:5, 3:7, or 3:8.In particular embodiments, which may combine the features of some or all of the above embodiments, generating the excitation light further includes, during a sub-interval within the interrogation time interval, simultaneously activating each light source associated with a group corresponding to the sub-interval. In particular embodiments, which may combine the features of some or all of the above embodiments, each band of excitation wavelengths includes one or more excitation wavelengths, and wherein each band of emission wavelengths includes one or more emission wavelengths. In particular embodiments, which may combine the features of some or all of the above embodiments, the sub-intervals within the interrogation time interval are non-overlapping in time. In particular embodiments, which may combine the features of some or all of the above embodiments, the distribution of the bands of excitation wavelengths into the plurality of groups is based on one or more of: (a) increasing a spectral separation of the bands of excitation wavelengths; (b) increasing a spectral separation of the bands of emission wavelengths; or (c) increasing a signal-to-noise ratio of detection.

[0021] In particular embodiments, which may combine the features of some or all of the above embodiments, a system includes eight wavelengths or bands of excitation wavelengths distributed into three groups. In particular embodiments, which may combine the features of some or all of the above embodiments, a first group of the three groups includes a first, a fourth, and a sixth wavelength or band of excitation wavelengths; wherein a second group of the three groups includes a second, a fifth, and an eighth wavelength or band of excitation wavelengths; and wherein a third group of the three groups includes a third and a seventh wavelength or band of excitation wavelengths. In particular embodiments, which may combine the features of some or all of the above embodiments, the wavelengths or bands of excitation wavelengths are numbered based on arranging the eight wavelength or bands of excitation wavelengths in order of increasing wavelength.

[0022] Systems for detecting presence of a fluorescent signal in a plurality of detection wells in a cartridge are disclosed. Systems of interest comprise a plurality of light sources operably linked to a plurality of detection wells, wherein each light source in the plurality of light sources is configured for emitting excitation light, comprising specified excitation wavelength band, in a sequential manner to sequentially illuminate a specified detection well of the plurality of detection wells; a photodetector (e.g., a single photodetector); a conduit for transmitting light emitted from the plurality of detection wells to the photodetector; and a processor for correlating the emitted light detected by the photodetector to the detection well emitting the light.As used herein, an “excitation wavelength” or an “emission wavelength” refers to a wavelength band which is made up of a specific set of wavelengths. Unless specified otherwise, as used herein, excitation wavelength or emission wavelength does not refer to a single wavelength and rather refers to a set of wavelengths, i.e., a specific and narrow wavelength band. For example, an excitation wavelength for exciting a fluorophore can comprise of a set of wavelengths suitable for exciting the fluorophore and can include a single wavelength that is the peak excitation wavelength and surrounding wavelengths. Surrounding wavelengths can range from ± 5 nm to ± 50 nm. A wavelength band for exciting a red fluorescent dye can include wavelengths ranging from 591 nm - 608 nm. In other words, an excitation wavelength for exciting a red fluorescent dye can include wavelengths ranging from about 591 nm - 608 nm. An excitation wavelength for exciting a red fluorescent dye can even include wavelengths ranging from 550 nm - 650 nm and the excitation wavelength can be filtered through a light filter, as described herein, to narrow the wavelengths transmitted to a detection well.

[0023] Systems of interest may comprise an excitation filter configured for transmitting the specified excitation wavelength or wavelength bands. Systems of interest may comprise a dichroic beam splitter configured for reflecting the specified excitation wavelength or wavelength bands to the conduit and for transmitting an emission wavelength band received from the conduit to the photodetector. The systems of interest may include a single photodetector for detecting the emission wavelength emitted from the plurality of detection wells. The systems of interest may include two or more photodetectors for detecting the emission wavelengths emitted from the plurality of detection wells.

[0024] The systems of interest may include a plurality of light pipes for transmitting an emission wavelength emitted from the plurality of detection wells to one or more photodetectors, e.g., a single photodetector or a plurality of photodetectors. The light pipes may include linear segments and curved segments. The linear segments and / or curved segments may be tapered to collimate the emission wavelength. The tapering may configure the light pipes for reduction of exit angle of light exiting the light pipes. Reducing the exit angle of light transmitted through the light pipes can increase collimation and focus the light. Thus, light exiting the light pipes and striking the photodetector is easily detected.

[0025] Devices for detecting the presence of fluorescent signals in a plurality of detection wells in a cartridge are disclosed. Devices of interest may comprise a cartridge staging region, and the system of the present disclosure. The cartridge staging region may be a slot sized to hold the cartridge.Semi-automated or automated methods of detecting fluorescence in a plurality of detection wells in a cartridge are disclosed. Methods of interest may comprise (a) generating an excitation light from a mthlight source of the plurality of light sources at a specified excitation wavelength or excitation wavelength band and illuminating a nth detection well of the plurality of detection wells and ceasing generating the excitation light; (b) transmitting the emission wavelength emitted from the nth detection well to one or more of the photodetectors; (c) detecting the emission wavelength on the one or more photodetectors; (d) correlating the emission wavelength detected by the one or more photodetectors to the nth detection well emitting the emission wavelength.

[0026] The method may comprise (e) repeating steps (a) to (d) a plurality of times to detect fluorescence in the plurality of detection wells. The method may comprise (e) repeating steps (a) to (d) a plurality of times, wherein during each repetition, the mthlight source emits light of a specified excitation wavelength or excitation wavelength band, which may be different in each repetition. The method may comprise (e) repeating steps (a) to (d) a plurality of times, to detect fluorescence in the plurality of detection wells, wherein during each repetition, the mthlight source emits light of a specified excitation wavelength or excitation wavelength band, which may be different in each repetition. The method may comprise (e) repeating steps (a) to (d) a plurality of times to detect fluorescence in the plurality of detection wells, followed by repeating steps (a) to (e) with a different excitation light from the mthlight source.

[0027] In particular embodiments, which may combine the features of some or all of the above embodiments, a method is disclosed for detecting presence of a plurality of fluorescent signals in a detection well, the method including: generating, by five light sources, excitation light comprising one or more wavelengths or bands of excitation wavelengths, wherein the one or more wavelengths or bands of excitation wavelengths are distributed into a plurality of groups such that each group is associated with one or more of the wavelengths or bands of excitation wavelengths; providing the excitation light to the detection well by sequentially illuminating the detection well over sub-intervals within an interrogation time interval, wherein each sub-interval is correlated with a respective group of the plurality of groups such that the illumination provided to the detection well during each sub-interval comprises one or more wavelengths or the bands of excitation wavelengths associated with the respective group; generating, by a sample disposed in the detection well based on illuminating the detection well, six wavelengths or bands of emission light over the interrogation time interval; and detecting, by one or more photodetectors, the six wavelengths or bands of emission light as the plurality of fluorescent signals over the interrogation time interval.In accordance with the disclosed subject matter, the one or more wavelengths or bands of emission light can include one or more wavelengths or bands of emission wavelengths respectively correlated with the one or more wavelengths or bands of excitation wavelengths provided to the detection well, and the emission light can be generated by the sample over the interrogation time interval. Generation can further include sequentially emitting, during each subinterval, the one or more wavelengths or bands of emission wavelengths correlated with the respective one or more wavelengths or bands of excitation wavelengths and associated with the respective group. Detecting the emission light can further include: separating the one or more wavelengths or bands of emission wavelengths generated by the sample by selectively filtering the emission light based on the plurality of groups; and directing the one or more wavelengths or bands of emission wavelengths of the emission light to a separate photodetector of the one or more photodetectors for detection, for example, a photodetector associated with each group, wherein each wavelength or band of emission wavelengths is detected as a corresponding fluorescent signal of the plurality of fluorescent signals.

[0028] In accordance with the disclosed subject matter, the method can include amplifying, by one or more amplifiers, the plurality of fluorescent signals detected by the one or more photodetectors; multiplexing, by a multiplexer, the plurality of amplified fluorescent signals; and converting, by an analog-to-digital converter, the multiplexed plurality of amplified fluorescent signals into a digital stream. Additionally or alternatively, the method can include normalizing or adjusting, by a normalization photodetector, an intensity of the excitation light generated by the one or more light sources based on sampling at least a portion of the excitation light.

[0029] In accordance with the disclosed subject matter, at least one group of the plurality of groups can include a plurality of wavelengths or a plurality of bands of excitation wavelengths. At least two of the plurality of wavelengths or the plurality of bands of excitation wavelengths associated with the at least one group can be simultaneously provided to the detection well during the sub-interval associated with the at least one group. The five light sources can be at least partially integrated into one or more light source components.

[0030] Generating the excitation light further can include sequentially activating the three groups of the five light sources over three sub-intervals within the interrogation time interval. Additionally or alternatively, generating the excitation light can include, during a sub-interval within the interrogation time interval, simultaneously activating each light source associated with a group corresponding to the sub-interval. Each band of excitation wavelengths can include one or more excitation wavelengths, and wherein each band of emission wavelengths can include oneor more emission wavelengths. In accordance with the disclosed subject matter the sub-intervals within the interrogation time interval can be non-overlapping in time.

[0031] Distribution of the one or more wavelengths or bands of excitation wavelengths into the plurality of groups can be based on one or more of: (a) increasing a spectral separation of the one or more wavelengths or bands of excitation wavelengths; (b) increasing a spectral separation of the bands of emission wavelengths; or (c) increasing a signal-to-noise ratio of detection. The plurality of groups can include three groups so that the five bands of excitation wavelengths are distributed into the three groups. A first group of the three groups can include a first wavelength or band of excitation wavelength; a second group of the three groups can include a third and fourth wavelength or band of excitation wavelengths; and a third group of the three groups can include a second and a fifth wavelength or band of excitation wavelengths. The wavelengths or bands of excitation wavelengths can be numbered based on arranging the five bands of excitation wavelengths in order of increasing wavelength. One or more steps of the method associated with detecting the presence of a plurality of fluorescence signals in a detection well can be applied to a plurality of detection wells.

[0032] Aspects of the present disclosure include an optical detection module.

[0033] BRIEF DESCRIPTION OF THE FIGURES FIG. 1A depicts a schematic of a system for detecting fluorescence in one or more detection wells, according to particular embodiments. In particular embodiments, ae direction of transmission of the excitation wavelength to one or more detection wells may be substantially perpendicular to the direction of transmission of the emission wavelength to a photodetector. In particular embodiments, there may be a 1: 1 correlation between the excitation source and the well. In particular embodiments, there may be a 1:1:1 correlation between the excitation source, the well, and the fluorescence detector.

[0034] FIG. 1B depicts a schematic of a system for detecting fluorescence in one or more detection wells, according to particular embodiments. In particular embodiments, at least two light sources can sequentially or simultaneously illuminate a detection well. In particular embodiments, there is a 2:1 correlation between the excitation source and the well.

[0035] FIG. 2A depicts a schematic of an exemplary system for detecting fluorescence in a plurality of wells, according to particular embodiments. In this system, the direction of transmission of the excitation wavelength to detection wells is substantially parallel to the direction of transmission of the emission wavelength to the photodetector.FIG.2B depicts a schematic of a system for detecting fluorescence, according to particular embodiments. In this embodiment, the direction of transmission of the excitation wavelength to detection wells is substantially parallel to the direction of transmission of the emission wavelength to the photodetector.

[0036] FIG. 2C depicts another schematic of the system for detecting fluorescence in a plurality of wells, according to particular embodiments. In this system, a single light source can emit light at two different excitation wavelengths.

[0037] FIG.2D depicts a schematic of a system for detecting fluorescence, according to particular embodiments. In this system, a single light source emits can light at two different excitation wavelengths.

[0038] FIG.2E depicts a schematic of a system for detecting fluorescence, according to particular embodiments.

[0039] FIG.2F depicts an exemplary scheme for distributing 8 wavelengths or bands of excitation light wavelengths into 3 groups and employing 8 exemplary dyes in a detection well, according to particular embodiments.

[0040] FIG. 2G schematically depicts dye absorption and dye emission spectra associated with the 8 dyes provided in a detection well and interrogated according to the exemplary scheme of FIG. 2F

[0041] FIG.2H depicts a schematic of a system for detecting fluorescence, according to particular embodiments.

[0042] FIG.3A depicts a schematic of a system for detecting fluorescence, according to particular embodiments.

[0043] FIG.3B depicts a schematic of a system for detecting fluorescence, according to particular embodiments.

[0044] FIGs. 4A and 4B show configuration of a plurality of light sources for use in systems for detecting fluorescence, according to particular embodiments. For example, and not by way of limitation, in the systems depicted in FIGs. 3A and / or 3B.

[0045] FIG. 5 shows plurality of light sources arranged in a concentric manner and aligned with light conduits arranged for directing light from the light sources to wells arranged in a linearconfiguration, according to particular embodiments. The individual light sources are depicted with a numeral to show an exemplary sequential illumination layout of the system.

[0046] FIG.6 shows an optical detection module, according to particular embodiments.

[0047] FIG.7 shows fluorescence measured using a system according to particular embodiments, such as that depicted in FIG. 1.

[0048] FIG. 8A shows a light pipe array having two ends, according to particular embodiments. At one end, the light pipes are attached to a linear support structure configured for positioning adjacent linearly arranged detection wells. At the other end the light pipes are attached to a circular support structure configured for positioning adjacent a single photodetector. The light pipes transmit light from a linear array of detection wells (not shown) to the single photodetector.

[0049] FIG.8B shows a light pipe with a linear segment that is reverse tapered by increasing the diameter of the light pipe from the point of entry towards point of exit from the light pipe, according to particular embodiments.

[0050] FIG. 8C shows a light pipe with reverse-tapered linear segments connected via curved segments, according to particular embodiments.

[0051] FIG. 8D depicts a schematic light pipe waveguide for relaying excitation light and returning emission light, according to particular embodiments.

[0052] FIGs. 9 A and 9B depict schematics of a system for detecting fluorescence, according to particular embodiments. A normalization photodetector is included to detect intensity of the excitation wavelength from the light sources and normalize either the intensity when a lack of uniform intensity is detected or normalize the corresponding emission wavelength to compensate for a lower intensity of the excitation wavelength from a light source.

[0053] FIG.10 shows a schematic of PCR cycles and timing of interrogation of fluorescent signal during extension phase in a PCR cycle, according to particular embodiments.

[0054] FIG. 11 shows an exemplary chart of the percent transmission of DBS1, DBS2, DBS3, the excitation filter, EFB1, EFB2, and EFB3of FIG. 2E, in accordance with the disclosed subject matter.DETAILED DESCRIPTION

[0055] The present disclosure provides devices, systems, and methods for detecting the presence of fluorescent signals in one or more detection wells. In particular embodiments, a device, system, and method may be useful for improving fluorescence detection and reducing optical components count, cost, size, etc. In particular embodiments, a device, system, and / or a method of present disclosure can be useful for collecting data from independent detection wells that may contain an analyte of interest, such as, nucleic acid indicative of presence of a pathogen in a sample being analyzed.

[0056] Before the present devices, systems, and methods are described in greater detail, it is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0057] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the present systems, devices and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the systems, devices and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the systems, devices and methods.

[0058] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating un-recited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present systems, devices and methods, representative illustrative systems, devices and methods are now described.The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0060] The term “comprising” is used herein as requiring the presence of the named component and allowing the presence of other components. The term “comprising” should be construed to include the term “consisting essentially of’ and “consisting of.” The “consisting essentially of’ allows the presence of the named component(s), along with other components which do not change the function / structure of the named component(s). The “consisting of” allows the presence of the named component(s).

[0061] Numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0062] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 2 ml to 10 ml” is inclusive of the endpoints, 2 ml and 10 ml, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0063] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of from about “2 to about 10” also discloses the range “from 2 to 10.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9- 4.1.

[0064] It should be noted that many of the terms used herein are relative terms. For example, the terms “top” and “bottom” are relative to each other in location and refer to surfaces where the top is always higher than the bottom relative to an absolute reference, i.e., the surface of the earth. The terms “inlet” and “outlet” are relative to a fluid flowing through them with respect to a given structure, e.g., a fluid flows through the inlet into the structure and flows out of the structure through the outlet. The terms “upwards” and “downwards” are also relative to an absolutereference; upwards is always against the gravity of the earth while downwards is always towards the gravity of the earth.

[0065] The term “parallel” should be construed in its lay sense of two surfaces that maintain a generally constant distance between them, and not in the strict mathematical sense that such surfaces will never intersect when extended to infinity.

[0066] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0067] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0068] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present device, systems and methods. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0069] SYSTEMS AND METHODS FOR DETECTING FLUORESCENCE

[0070] The present disclosure provides systems for detecting presence of a fluorescent signal in one or more detection wells. In particular embodiments, a plurality of detection wells may be provided in a cartridge.

[0071] In particular embodiments, a system may comprise a plurality of light sources operably linked to the plurality of detection wells, wherein individual light sources of the plurality of light sources are configured for emitting excitation light at a specified excitation wavelength in asequential manner to illuminate a specified detection well of the plurality of detection wells. In particular embodiments, a system may also comprise one or more photodetectors; a conduit for transmitting an emission wavelength emitted from the plurality of detection wells to one of the photodetectors; and / or a processor for correlating the emission wavelength detected by the photodetector to the detection well emitting the emission wavelength.

[0072] In particular embodiments, a system may comprise one or more light sources operably linked to one or more detection wells in any suitable combination. In particular embodiments, each light source in a plurality of light sources may be configured for emitting excitation light at a specified excitation wavelength or a band in a sequential manner to illuminate a specified detection well; one or more photodetectors; one or more conduits for transmitting emission wavelengths emitted from the plurality of detection wells to a photodetector; and a processor for correlating the emission wavelength detected by the one or more photodetector(s) to the one or more detection well(s) emitting the one or more emission wavelength(s). The processor may correlate the emission wavelength detected by the photodetector to the detection well emitting the emission wavelength by matching information regarding the light source activated to illuminate the detection well.

[0073] The plurality of light sources may be arranged on a single plane, e.g., a single vertical or horizontal plane. The light sources of the plurality of light sources may be arranged on the single plane in a linear row. The light sources of the plurality of light sources may be arranged in a circular configuration on a single plane.

[0074] The plurality of detection wells may be arranged on a single plane. The plurality of detection wells may be arranged in a linear row. The plurality of detection wells may be arranged in a circular configuration. The plurality of detection wells may be arranged in a linear row or in a circular configuration on a single plane.

[0075] The plurality of light sources may comprise “m” sources that are configured to illuminate “n” detection wells, wherein ration of m:n is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:2, 3:1, 3:4, 3:5, 4:1, 4:3, 4:5, 5:1, 5:2, 5:3, or 5:4. The plurality of light sources may comprise at least 8 light sources that are configured to illuminate at least 8 detection wells; at least 12 light sources that are configured to illuminate at least 12 detection wells; at least 16 light sources that are configured to illuminate at least 16 detection wells, wherein there is a 1 to 1 co-relation. The plurality of light sources may comprise at least 8 light sources that are configured to illuminate at least 16 detection wells; at least 12 light sources that are configured to illuminate at least 24 detection wells; at least16 light sources that are configured to illuminate at least 32 detection wells, wherein there is a 1 to 2 co-relation.

[0076] The plurality of light sources may comprise at least 8 light sources that are configured to illuminate at least 4 detection wells; at least 12 light sources that are configured to illuminate at least 6 detection wells; at least 16 light sources that are configured to illuminate at least 8 detection wells, wherein there is a 2 to 1 co-relation. The plurality of light sources may comprise at least 32 light sources configured to illuminate at least 16 detection wells, wherein there is a 2 to 1 corelation.

[0077] The plurality of light sources may be arranged in a linear array, where a mthlight source illuminates the nth detection well, wherein m:n is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:2, 3:1, 3:4, 3:5, 4: 1, 4:3, 4:5, 5: 1, 5:2, 5:3, or 5:4. The plurality of light sources may be arranged in pairs in a linear array, where a first light source and a second light source in each pair illuminates the same detection well and wherein the first light source is capable of emitting excitation light at an excitation wavelength different from excitation light emitted by the second light source.

[0078] The light source in the plurality of light sources may be a light-emitting diode (LED), wherein the LED emits excitation light at one or more excitation wavelengths. LEDs are made from a variety of inorganic semiconductor materials which enable emission of light at one or more selected colors. LEDs capable of emitting light having wavelengths ranging from about 275 to 950 nm are available. LEDs manufactured from semiconductor material Indium nitride (InN) emit blue and green light. LEDs manufactured from semiconductor indium gallium nitride (InGaN) emit blue, green and ultraviolet light. LEDs manufactured from semiconductor Gallium(III) nitride (GaN) emit ultraviolet light. LEDs manufactured from Gallium arsenide (GaAs) emit red and infrared light. Semiconductor materials may be doped and / or manufactured using combination of elements (e.g., Indium, Gallium, Phosphorus, and / or Arsenic) to produce alloys that enable emission of light of specific wavelengths.

[0079] A light source in the plurality of light sources may be capable of emitting at least two different excitation lights having different excitation wavelengths or wavelength bands. The light source may first emit light at a first excitation wavelength or wavelength band to illuminate a detection well. The light source may then emit light at a second excitation wavelength or wavelength band and illuminate the same detection well. If the detection well includes two different fluorophores, both fluorophores can be detected in the same detection well using a single light source.A light source in the plurality of light sources may be capable of emitting at least two different excitation lights having different excitation wavelengths or wavelength bands. The light source may emit light at a first excitation wavelength or wavelength band and illuminate a first detection well. The light source may then emit light at the first excitation wavelength or wavelength band and illuminate a second detection well. The light source can emit light at a second excitation wavelength or wavelength band and illuminate the same first detection well. The light source can then emit light at the second excitation wavelength and illuminate the same second detection well.

[0080] Each light source may be capable of emitting excitation lights at four or more different excitation wavelengths or excitation wavelength bands. The conduit for transmitting an emission wavelength emitted from the plurality of detection wells to the photodetector may comprise a plurality of light conduits each configured to transmit light from one of the plurality of detection wells. The light conduits may comprise of a plurality of light pipes. The light conduits may comprise of a plurality of optical fibers. The light conduits may comprise one or more mirrors. The focusing optic(s) may be positioned between the light conduits and the photodetector(s) to collimate light exiting from the light conduits before the light impinges on the photodetector(s). The light conduits may be configured to collimate the light traversing therethrough and focusing optic(s) is not positioned between the light conduits and the photodetector(s) to collimate light exiting from the light conduits before the light impinges on the photodetector(s).

[0081] The systems described herein may include a single photodetector. The single photodetector may be used to detect the emission from a detection well upon illumination by a light source. When a single photodetector is used, a light source may illuminate a first detection well, followed by illumination of a second detection well and so on and a processor may decipher which detected signal emanated from which detection well based on known sequence of illumination by the light source. Similarly, a first light source may illuminate a first detection well, followed by illumination of a second detection well by a second light source and so on and a processor may decipher which detected signal emanated from which detection well. Similarly, a first light source may illuminate a first detection well, followed by illumination of the first detection well by a second light source, followed by illumination of the first detection well by a third light source, and so on and a processor may decipher which detected signal corresponds to which light source. Distinguishing which detected signal corresponds to which light source may utilize the subinterval of when the detection well is illuminated with the excitation light.Similarly, a first light source may illuminate a first detection well, followed by illumination of the first detection well by a second light source, followed by illumination of the first detection well by a third light source, and so on and a first light source may illuminate a second detection well, followed by illumination of the second detection well by a second light source, followed by illumination of the second detection well by a third light source, and so on and a processor may decipher which detected signal corresponds to which light source and which well. Distinguishing which detected signal corresponds to which light source may utilize the sub-interval of when the detection first and the second well are illuminated with the excitation light.

[0082] In some embodiments, each of the detection wells comprise a sample which may include a positive control having a first fluorescent profile and fluorescent molecules indicative of presence of an analyte. The analyte may be a molecule that is bound to a fluorescent molecule (e.g., a fluorescently tagged antibody, a fluorescent oligonucleotide) or has incorporated fluorescent molecule, e.g., fluorescent bases. The fluorescent molecules may be selected from FAM, ROX, Alexa, rhodamine, etc. Additional fluorescent molecules that can be used in the systems and methods disclosed herein are known in the art and use of such fluorescent molecules is within the purview of the disclosure. In specific embodiments, the fluorescent molecules are large stokes shift fluorescent molecules, which are fluorescent molecules exhibit a large gap between the excitation and the emission maxima, for example, a gap of 80 nm or more, such as 100 nm or more, 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, or 150 nm or more. Certain examples of large stokes shift dyes include Lucifer Yellow (disulfonylated imide of 4-aminaphthalene 1,8-dicarboxylic acid), APTS (1-aminopyrene-3,6,8-trisulfonate), NBD-X (N-alkyl-4- amino-7-nitrobenz-2-oxa-l,3-diazol), propidium iodide, ATTO465, ATTO430LS, and ATTO490LS. Additional examples of large stokes shift dyes are described in Sednev et al. (2015), Methods and Applications in Fluorescence, 3:042004, which is herein incorporated by reference in its entirety.

[0083] In some embodiments, each of the light sources in the plurality of light sources are configured for emitting the excitation wavelength in a first direction such that the excitation wavelength enters at a bottom region of the detection wells. In some examples, the light sources are placed below the bottom region of the detection wells. In some examples, one light source is positioned below two detection wells such that the single light source emits excitation light at one excitation wavelength to illuminate a first detection well; and then the same light source emits excitation light at the same or another excitation wavelength to illuminate a second detection well. Thus, in certain embodiments, the light source emits two different excitation wavelengths. In someexamples, the light source may emit four different excitation wavelengths and illuminate four detection wells.

[0084] In some examples, one light source is positioned below one detection well such that the number of detection wells is equal to the number of light sources. In some examples, two light sources are positioned below one detection well such that the number of light sources is double the number of detection wells. In this embodiment, the two light sources emit two different excitation wavelengths. For example, one excitation wavelength may be for exciting a fluorescence molecule indicative of the presence of an analyte of interest in a sample and the other excitation wavelength may be for exciting a fluorescence molecule that is a positive control that indicates that the sample is present and / or substantially equal volumes of samples are present in the plurality of detection wells. In some examples, a collimating lens is positioned between the light source and the detection wells.

[0085] A light filter for allowing selected excitation wavelengths to enter the detection wells may be positioned between the light source and the detection wells. This light filter is referred to as an excitation filter. A light filter for allowing selected emission wavelengths to reach the photodetector may be positioned between the detection wells and the photodetector. This light filter is referred to as an emission filter. The system may comprise an excitation filter and an emission filter. The system may comprise multiple excitation filters. For example, each light source may be associated with an excitation filter such that light emitted from the light source passes through the excitation filter before striking a detection well. Any suitable wavelength filter may be used as a filter in the systems disclosed herein. For example, traditional Fabry-Perot filters may be used in the systems disclosed herein. Multiband filters that transmit more wavelengths than the wavelengths absorbed may be used in the disclosed systems to ensure that a range of excitation lights can be transmitted to the detection wells and a range of light emitted from the detection wells can be relayed to the photodetector may be used in the disclosed systems. A multiband filter for use as an excitation filter and a multiband filter used as an emission filter may be selected to ensure little or no overlap on the wavelengths absorbed and emitted by the filter. Thus, an excitation filter is selected such that it emits a light having a wavelength band that excites the fluorophore being detected and ensures that the light does not include wavelengths that overlap with the light emitted from the excited fluorophore. Correspondingly, an emission filter is selected which does not allow the excitation light to reach the photodetector (by absorbing the excitation light) while allowing the light emitted by the excited fluorophore to reach the photodetector.The conduit for transmitting emission wavelength emitted from the plurality of detection wells to the photodetector may transmit light in a second direction which is orthogonal to the first direction in which the light sources emit the light. An example of such an orthogonal architecture is depicted in FIGs. 1A and IB.

[0086] The present disclosure also includes a second system for detecting presence of a fluorescent signal in a plurality of detection wells in a cartridge, the system comprising: a plurality of light sources arranged on a single plane; an excitation filter positioned adjacent to the plurality of light sources; a dichroic beam splitter arranged at an angle ranging from 30-60 degrees with reference to the single plane; a conduit for interrogating one of a plurality of predetermined detection wells; one or more photodetectors; and a processor for correlating light detected by one of the photodetectors to one of the plurality of detection wells; wherein: each light source in the plurality of light sources is configured for emitting an excitation light at a specified excitation wavelength in a sequential manner to illuminate a specified detection well of the plurality of detection wells such that one detection well is illuminated at a time; the excitation filter is configured for transmitting the specified excitation wavelength towards the dichroic beam splitter; the dichroic beam splitter is configured for reflecting the specified excitation wavelength to the conduit and for transmitting an emission wavelength received from the conduits to the one of the photodetectors; the photodetector is configured for detecting the emission wavelength received from the detection well; and the processor correlates the emission wavelength detected by photodetector to the detection well emitting the emission wavelength.

[0087] In certain aspects, the system comprises: a plurality of light sources arranged on a single plane; an excitation filter positioned adjacent to the plurality of light sources; a dichroic beam splitter arranged at an angle ranging from 30-60 degrees with reference to the single plane; a conduit for interrogating one of a plurality of predetermined detection wells; a single photodetector; and a processor for correlating light detected by the photodetector to one of the plurality of detection wells; wherein: each light source in the plurality of light sources is configured for emitting an excitation light at a specified excitation wavelength in a sequential manner to illuminate a specified detection well of the plurality of detection wells such that one detection well is illuminated at a time; the excitation filter is configured for transmitting the specified excitation wavelength towards the dichroic beam splitter; the dichroic beam splitter is configured for reflecting the specified excitation wavelength to the conduit and for transmitting an emission wavelength received from the conduit to the photodetector; the photodetector is configured for detecting the emission wavelength received from the predetermined detection well;and the processor correlates the emission wavelength detected by photodetector to the detection well emitting the emission wavelength.

[0088] The conduit may comprise a plurality of light conduits each configured to transmit light to one of the plurality of detection wells. The conduit may comprise a dynamic steerable mirror configured to sequentially reflect light to one of the plurality of detection wells.

[0089] The light sources of the plurality of light sources may be arranged in a circular configuration. The light sources of the plurality of light sources may be arranged in a concentric configuration. An outer circle of the concentric configuration may comprises at least 12 light sources and the inner circle of the concentric configuration comprises at least 4 light sources. The photodetector may be a single photodetector.

[0090] The photodetector may be positioned on a plane that is substantially perpendicular to the single plane on which the light sources are arranged. The photodetector may be positioned on a plane substantially parallel to the single plane on which the light sources are arranged.

[0091] FIG. 1A represents a schematic of a system for detecting fluorescence in one or more detection wells, according to particular embodiments. In particular embodiments, multiple detection wells may be positioned in a row in a cartridge. In particular embodiments, such a system may include a plurality of light sources arranged on a single plane (not shown) in a linear configuration such that at least one light source is located below an area where a detection well is positioned. In this non-limiting example, the light sources (i.e., excitation source n and excitation source n+1) can be placed adjacent to each other such that each of these light sources transmit light into the corresponding detection wells (excitation source n transmits light to well n and excitation source n+1 transmits light to well n+1). In particular embodiments, a plurality of light sources may be arranged on a single plane in a linear configuration. Not shown in the figure is a circuit for controlling the light sources. In particular embodiments, a circuit can be configured to emit light from each light source into corresponding wells below which the light sources are positioned. In particular embodiments, each light source can be configured for emitting light at a specified excitation wavelength, for example, to illuminate a specified detection well. In particular embodiments, light sources may be activated simultaneously and / or sequentially with other light sources to emit the excitation light. As shown herein by way of example and not limitation, two photodetectors may be present (detector n and detector n+1). Also shown by way of example and not limitation are excitation filters for controlling the wavelength of light transmitted from the excitation source to the detection well.FIG. IB represents schematic of a system for detecting fluorescence in one or more detection wells, according to particular embodiments. The detection wells are positioned in a row in the cartridge. This system includes a plurality of light sources arranged on a single plane in a linear configuration such that at least two light sources are located below each detection well. In this example, four light sources (nx, ny, (n+l)x and (n+l)y) are arranged linearly and placed immediately adjacent to each other such that each of these light sources transmit light into the corresponding detection wells (i.e., excitation sources nx and ny illuminate well n, excitation sources (n+l)x and (n+l)y) illuminate well n+1). Light source nx may first illuminate detection well (n), followed by light source ny illuminating detection well (n), followed by light source (n+l)x illuminating detection well (n+1) and then light source (n+l)y illuminates sample well (n+1). In other words, the light sources turn on in a sequential manner. The system may be configured to sequentially illuminate the wells and repeat the sequential illumination a plurality of times. Not shown in the figure is a circuit for controlling the light sources. Each light source in the plurality of light sources is configured for emitting light at a specified excitation wavelength in a sequential manner to illuminate a specified detection well of the plurality of detection wells. Light sources nx and (n+l)x may emit light of the same wavelength. Light sources ny and (n+l)y may emit light of the same wavelength. Light sources nx and (n+l)x may emit light of the same wavelength and light sources ny and (n+l)y may emit light of the same wavelength, which wavelengths are different from each other. The light sources nx and ny may be configured for exciting different fluorophores that may be present in a single detection well. This system include a photodetector for each detection well. Two photodetectors are depicted (detector n and detector n+1). Photodetector (n) sequentially detects fluorescence from detection well (n) sequentially illuminated by light source nx and ny. Detector (n+1) sequentially detects fluorescence from detection well (n+1) sequentially illuminated by light source (n+l)x and (n+l)y. Also shown are excitation filters for transmitting light at a specified excitation wavelength to illuminate a specified detection well and emission filters for transmitting light to the photodetectors.

[0092] FIGs. 1A and IB depict an orthogonal system in which the plane on which the light sources are located is orthogonal to the plane on which the detector are located.

[0093] FIG. 2A represents a schematic of an exemplary system for detecting fluorescence, according to particular embodiments of the present disclosure. This schematic of the system includes a plurality of light sources arranged on a single plane. For example, the system includes a light source (m) and another light source (m+1). Each light source in the plurality of light sources is configured for emitting light at a specified excitation wavelength in a sequential manner toilluminate a specified detection well of the plurality of detection wells. For example, light source (m) emits light at specified excitation wavelength to illuminate detection well (n). Similarly, light source (m+1) emits light at specified excitation wavelength to illuminate detection well (n+1). The system may include one or more photodetectors. In this example, a single photodetector is shown and the photodetector is on a plane substantially perpendicular to the single plane on which the light sources are arranged. An excitation filter is shown to be arranged on the same plane and is configured for transmitting the specified excitation wavelength towards the dichroic beam splitter. A dichroic beam splitter is shown and is arranged at an angle ranging from 30-60 degrees with reference to the single plane on which the plurality of light sources are arranged. A focus lens and an emission filter is positioned between the dichroic beam splitter and the photodetector. The dichroic beam splitter is configured for reflecting the specified excitation wavelengths from the light sources to the detection wells and for transmitting emission wavelengths received from the detection wells to the photodetector. In use, light source (m) emits excitation light at a specified excitation wavelength towards the excitation filter which transmits the light towards the dichroic beam splitter. The dichroic beam splitter reflects the light to the detection well (n) and illuminates it. The emission light illuminated from detection well (n) is transmitted through the dichroic beam splitter and is received by the photodetector. Similar steps of fluorescence detection occur for light source (m+1).

[0094] FIG. 2C represents a system for detecting fluorescence, according to particular embodiments of the present disclosure. This schematic of the system includes a plurality of light sources arranged on a single plane. For example, the system includes a light source (n) and another light source (n+1). Each light source in the plurality of light sources is configured for emitting light at a specified excitation wavelength in a sequential manner to illuminate a specified detection well of the plurality of detection wells. For example, light source (n) emits light at excitation wavelengths (λ1) and (λ2) in sequential manner to illuminate detection well (n). Similarly, light source (n+1) emits light at excitation wavelengths (λ1) and (λ2) in sequential manner to illuminate detection well (n+1). The system may include one or more photodetectors. In this example, a single photodetector is shown and the photodetector is on a plane substantially perpendicular to the single plane on which the light sources are arranged. An excitation filter is shown to be arranged on the same plane and is configured for transmitting the specified excitation wavelength towards the dichroic beam splitter. A dichroic beam splitter is shown and is arranged at an angle ranging from 30-60 degrees with reference to the single plane on which the plurality of light sources are arranged. A focus lens and an emission filter are positioned between the dichroic beam splitter and the photodetector. The dichroic beam splitter is configured for reflecting the specifiedexcitation wavelengths from the light sources to the detection wells and for transmitting emission wavelengths received from the detection wells to the photodetector. In use, light source (n) emits excitation light at the excitation wavelength (XI) towards the excitation filter which transmits the light towards the dichroic beam splitter. The dichroic beam splitter reflects the light to the detection well (n) and illuminates it. The emission light illuminated from detection well (n) is transmitted through the dichroic beam splitter and is received by the photodetector. In sequential manner, light source (n) then emits excitation light at the excitation wavelength ( 2) towards the excitation filter which transmits the light towards the dichroic beam splitter. The dichroic beam splitter reflects the light to the detection well (n) and illuminates it. The emission light illuminated from detection well (n) is transmitted through the dichroic beam splitter and is received by the photodetector. Similar steps of fluorescence detection occur for light source (n+1).

[0095] FIGs. 2B and 2D depict systems in which the light sources and the photodetector can be positioned in the same plane.

[0096] FIG. 3A represents a schematic of a system for detecting fluorescence, according to particular embodiments of the present disclosure. This schematic of the system includes a plurality of light sources arranged in a circular configuration on a single plane. Each light source in the plurality of light sources is configured for emitting light at a specified excitation wavelength in a sequential manner to illuminate a specified detection well of the plurality of detection wells. In this example, the light sources are light emitting diodes (LEDs) arranged in a collimator ring. A single photodetector is shown and a conduit for transmitting an emission wavelength to the single photodetector is shown. The conduit is in the form of light pipes which enable transmission of emission wavelength emitted from the plurality of detection wells to the single photodetector. In this schematic, each detection well is operably connected to a light pipe such that the light pipe transmits emission wavelengths originating from the detection well. An emission focus lens is positioned between the light pipes and the single photodetector. A dichroic beam splitter is shown and is arranged at an angle ranging from 30-60 degrees with reference to the single plane on which the plurality of light sources are arranged. The dichroic beam splitter is configured for reflecting the specified excitation wavelength from the light sources to the light pipes and for transmitting emission wavelengths received from the light pipes to the photodetector. In this schematic, each detection well is operably connected to a light pipe such that the light pipe transmits emission wavelengths originating from the detection well. The distance between the detection wells and opening of the light pipes can be any suitable distance. A suitable distance can include 10 mm, 5 mm, 3 mm, 2 mm, 1 mm, or 0.5 mm. An emission focus lens is positioned between the light pipesand the single photodetector. In this system, the direction of transmission of the excitation wavelength is orthogonal to the direction of transmission of the emission wavelength and the plane on which the LEDs are located is perpendicular to the plane on which the photodetector is located.

[0097] The system of the present disclosure may further comprise a catadioptric system. The catadioptric system may comprise of an emission focus lens and a parabolic focus mirror, catadioptric system optionally comprises of a parabolic folding mirror. Parabolic folding mirror is useful for aligning the return beam to be coplanar with the excitation sources and reduce complexity of the systems as described in above section.

[0098] The conduit may comprise light conduits or steerable mirror. In some embodiments, the light conduits comprise of a plurality of light pipes. In some embodiments, the light conduits comprise of a plurality of optical fibers. In some embodiments, the light conduits comprise of a focusing optic. In some embodiments, the light conduits comprise of a mirror.

[0099] In some embodiments, the system of the present disclosure further comprises an element proximal to the light sources collimating light emitted from the light sources. In some embodiments, the element generates multiple collimated beams of light to the dichroic beam splitter to launch the filtered reflected light into the associated light conduits. In some embodiments, the element is a collimating lens ring. In some embodiments, the element is a collimating lens.

[0100] Tn some embodiments, the system of the present disclosure further comprises an element for collimating light exiting from the detection wells before the light enters the light conduits. For example, a collimating lens may be positioned between the detection wells and the light conduits. In some embodiments, the system of the present disclosure further comprises an element for collimating light exiting the light conduits before the light is detected by the photodetector. For example, a collimating lens may be positioned between the light conduits and the photodetector. The light pipe may be configured to collimate light traversing through the light pipe. In these embodiments, a collimating lens need not be positioned between the light pipes and the photodetector. Light collimating light pipes improve simplicity of the system and reduce cost. A light pipe for collimating light entering the light pipe may include a length between an entrance point at which light enters the light pipe and an exit point at which the light exits the light pipe. The light pipe may include at least one linear segment and at least one curved segment along the length, a tapered shape wherein a diameter of the light pipe changes, wherein the at least one linear segment comprises the tapered shape and / or wherein the at least one curved region comprises thetapered shape. The light pipe may include at least two linear regions connected by a curved region. The light pipe may include at least three linear regions and at least two curved regions, wherein the linear regions and curved regions alternate.

[0101] A system of light pipes for collimating light entering the light pipes is also disclosed. The system may include a plurality of light pipes, where each light pipe is the light pipe as described herein. The system may include a first planar support comprising a plurality of openings for securing the entrance points of the light pipes and a second planar support comprising a plurality of openings for securing the exit points of the light pipes. In some embodiments, the first support is rectangular in shape and the entrance points of the light pipes are arranged in a linear row across the support and match the layout of the detection wells and the second support is circular in shape and the exit points of the light pipes are arranged in a concentric shape.

[0102] The light pipes include a lumen that may include a coating to increase the amount of reflection of light waves travelling in the light pipes. In other embodiments, a coating is not included. The light pipes may be made from any suitable material. In some embodiments, the light pipe is made from a thermoplastic polymer. In some embodiments, the thermoplastic polymer is polycarbonate.

[0103] FIG. 2B represents a schematic of a system for detecting fluorescence, according to particular embodiments of the present disclosure. This schematic of the system includes a plurality of light sources arranged on a single plane. For example, the system includes a light source (m) and another light source (m+1). Each light source in the plurality of light sources is configured for emitting light at a specified excitation wavelength in a sequential manner to illuminate a specified detection well of the plurality of detection wells. For example, light source (m) emits light at specified excitation wavelength to illuminate detection well (n). Similarly, light source (m+1) emits light at specified excitation wavelength to illuminate detection well (n+1). The system may include one or more photodetectors. In this example, a single photodetector is shown and the photodetector is on a plane substantially parallel to the single plane on which the light sources are arranged. An excitation filter is shown to be arranged on the same plane and is configured for transmitting the specified excitation wavelength towards the dichroic beam splitter. A dichroic beam splitter is shown and is arranged at an angle ranging from 30-60 degrees with reference to the single plane on which the plurality of light sources are arranged. A focus lens and an emission filter is positioned between the dichroic beam splitter and the photodetector. The dichroic beam splitter is configured for reflecting the specified excitation wavelengths from the light sources to the detection wells and for transmitting emission wavelengths received from the detection wellsto the photodetector. An emission focus lens is shown and a folding mirror (as shown in FIG. 2D) can be used. An emission filter is shown to be arranged on the same plane and is configured for transmitting the specified emission wavelength towards the photodetector. In use, light source (m) emits excitation light at a specified excitation wavelength towards the excitation filter which transmits the light towards the dichroic beam splitter. The dichroic beam splitter reflects the light to the detection well (n) and illuminates it. The emission light illuminated from detection well (n) is transmitted through the dichroic beam splitter and towards the focus lens. Through the focus lens, the emission light can travel toward the folding mirror (if used and as shown in FIG. 2D) and is directed to the emission filter. The emission light is then received by the photodetector. Similar steps of fluorescence detection occur for light source (n+1).

[0104] FIG. 2D represents a schematic of a system for detecting fluorescence, according to particular embodiments of the present disclosure. This schematic of the system includes a plurality of light sources arranged on a single plane. For example, the system includes a light source (n) and another light source (n+1). Each light source in the plurality of light sources is configured for emitting light at a specified excitation wavelength in a sequential manner to illuminate a specified detection well of the plurality of detection wells. For example, light source (n) emits light at excitation wavelengths (λ1) and (λ2) in sequential manner to illuminate detection well (n). Similarly, light source (n+1) emits light at excitation wavelengths (λ1) and (λ2) in sequential manner to illuminate detection well (n+1). The system may include one or more photodetectors. In this example, a single photodetector is shown and the photodetector is on a plane substantially parallel to the single plane on which the light sources are arranged. An excitation filter is shown to be arranged on the same plane and is configured for transmitting the specified excitation wavelength towards the dichroic beam splitter. A dichroic beam splitter is shown and is arranged at an angle ranging from 30-60 degrees with reference to the single plane on which the plurality of light sources are arranged. A focus lens and an emission filter is positioned between the dichroic beam splitter and the photodetector. The dichroic beam splitter is configured for reflecting the specified excitation wavelengths from the light sources to the detection wells and for transmitting emission wavelengths received from the detection wells to the photodetector. An emission focus lens and a folding mirror is shown. An emission filter is shown to be arranged on the same plane and is configured for transmitting the specified emission wavelength towards the photodetector. In use, light source (n) emits excitation light at the excitation wavelength (λ1) towards the excitation filter which transmits the light towards the dichroic beam splitter. The dichroic beam splitter reflects the light to the detection well (n) and illuminates it. The emission light illuminated from detection well (n) is transmitted through the dichroic beam splitter and towards the focuslens. Through the focus lens, the emission light travels toward the folding mirror and is directed to the emission filter. The emission light is then received by the photodetector. In sequential manner, light source (n) then emits excitation light at the excitation wavelength (λ2) towards the excitation filter which transmits the light towards the dichroic beam splitter. The dichroic beam splitter reflects the light to the detection well (n) and illuminates it. The emission light illuminated from detection well (n) is transmitted through the dichroic beam splitter and towards the focus lens. Through the focus lens, the emission light travels toward the folding mirror and is directed to the emission filter. The emission light is then received by the photodetector. Similar steps of fluorescence detection occur for light source (n+1). In particular embodiments, particular photodetectors may be responsive to a broad band of radiation. By way of example and not limitation, particular Silicon-based photodetectors may be responsive between about 350-400 nm and about 1050-1150 nm. However, particular constraints may require utilization of a compressed spectral space to provide multiple bands of excitation wavelengths to a detection well, and receive correspondingly correlated bands of emission wavelengths from a sample provided in detection well. By way of example and not limitation, availability and / or suitability of particular dyes to an application may constrain an available spectral space for interrogation to a narrow or compressed subset of the spectral space in particular embodiments. Methods, systems, and devices disclosed herein may be used to enable use of multiple bands of wavelengths in a spectral, including but not limited to a particularly compressed spectral space, while minimizing intra-channel bleed over and / or cross-talk between bands, and / or while improving signal-to-noise ratios of detection.

[0105] FIG.2E depicts a schematic of a system for detecting fluorescence, according to particular embodiments. In particular embodiments, a detection well may be interrogated by multiple bands of excitation light. By way of example and not limitation, a multi-channel PCT detection system may comprise methods, systems, and / or devices disclosed herein where a detection well may be interrogated by multiple bands of excitation light. By way of example and not limitation, a detection well may be interrogated by 2, 4, or 8, or 16 bands of excitation light in particular embodiments.

[0106] By way of example and not limitation, particular embodiments will be described further herein comprising interrogation of a detection well with particular numbers of bands of excitation wavelengths (e.g., eight bands) distributed across particular numbers of groups (e.g., three groups). It will be appreciated that other combinations of parameters, such as bands, numbers of bands, and / or number of groups, are possible and fully contemplated herein, and that the concepts, methods, systems, and devices are accordingly extensible to any range of parameters. Specificparameters are selected herein only to provide a better understanding as non-limiting illustrative examples.

[0107] In particular embodiments, a system may be configured to provide 8 bands of excitation wavelengths from one or more light sources to a detection well. In particular embodiments, a suitable light source may comprise 8 distinct narrow-band LED sources. In particular embodiments, the light sources associated with providing the 8 bands may be located close to each other. In particular embodiments, the light sources associated with providing the 8 bands may be integrated on a multi-channel substrate, such as an 8 semiconductor substrate die. By way of example and not limitation, an 8 semiconductor substrate die may be placed in a suitable integrated circuit, such as a 9-pin monolithic microwave integrated circuit (MMIC) having a common connection to one terminal (e.g., cathodes) so that the remaining 8 terminals may be used to individually address each band and / or color of excitation wavelength.

[0108] In particular embodiments, a detection well such as schematically illustrated in FIG. 2E and configured to receive n bands of excitation wavelengths may be further configured to support n reactions, such as for amplification associated with PCR thermal cycles. By way of example and not limitation, a detection well configured to receive 8 bands of excitation wavelengths may support 8 reactions. By way of example and not limitation, 8 reactions in such a detection well may be associated with 40 PCR thermal cycles, for e.g., nominally at 15 seconds duration for each cycle. FIG. 10 schematically illustrates exemplary PCR cycles and associated timing of interrogation of corresponding fluorescent signals, according to particular embodiments. In particular embodiments, a system such as depicted in FIG.2E may be referred to as a multi-color detection module, wherein the detection well is disposed outside of the detection module and interfacing with it.

[0109] In particular embodiments, a first conduit may be provided to relay or direct the excitation light from one or more light sources to the detection well. In particular embodiments, a light pipe waveguide may be configured as the first conduit to relay the excitation light from the light sources to the detection well. In particular embodiments, a second conduit may be provided to relay or direct the emission light generated by a sample away from the detection well. In particular embodiments, a light pipe waveguide may be configured as the second conduit to relay the excitation light away from the detection well. In particular embodiments, the same waveguide may be used as the first conduit and the second conduit. FIG. 8D depicts a schematic light pipe waveguide for relaying excitation light and returning emission light, according to particular embodiments. By way of example and not limitation, the waveguide depicted in FIG. 8D isconfigured to relay 16 bands of light, comprising 8 bands of excitation light and 8 bands of emission light, respectively to and from the detection well. FIG.8D further schematically depicts model ray traces of light corresponding to the plurality of bands of light relayed therewithin. In particular embodiments, a light pipe waveguide may separately or additionally be significantly shortened relative to typical implementations. By way of example and not limitation, a light pipe waveguide may have a length between 15-35 mm, e.g., 25 mm, having a collimation half angle between 6-12 degree, e.g., 9 degrees. In particular embodiments, a light pipe waveguide may separately or additionally be configured in a linear and / or colinear configuration. In particular embodiments, a linear and / or colinear configuration of a light pipe waveguide may be permitted by a reduced complexity of relaying light beams to a single detection well. In particular embodiments, a colinear architecture relative to alternatives can be more highly manufacturable, and / or its size can be much more compact.

[0110] In particular embodiments, the detection well may be provided in an amplification chamber. In particular embodiments, the amplification chamber may be consumable. In particular embodiments, the amplification chamber may be reusable. In particular embodiments, the amplification chamber may comprise one or more other detection wells, wherein one or more of the other detection wells may respectively be configured for interrogation by multiple bands of excitation wavelengths, as may be understood based on the embodiments disclosed herein.

[0111] By way of example and not limitation, each band of light may be separately or additionally referred to herein as a color of light, and / or as a channel of light. In particular embodiments, such methods, devices, and / or systems disclosed wherein a detection well is interrogated by multiple bands of excitation light may be further extended to systems comprising multiple detection wells. It will be appreciated that this disclosure contemplates the adaptation and / or combination of any suitable methods, devices, and / or systems, such as those disclosed elsewhere herein, toward methods and systems wherein at least one detection well may be interrogated by multiple bands of excitation light.

[0112] In particular embodiments, the multiple bands of excitation wavelengths provided by the one or more light sources may be incident on an excitation filter, such as depicted in FIG. 2E by way of example and not limitation. In particular embodiments, the excitation filter may comprise interference filter layers configured to pass the multiple bands, e.g., 8 narrow bands, of excitation wavelengths.In particular embodiments, at least a portion of the excitation light may be sampled by a normalization photodetector. By way of example and not limitation, such as illustrated in FIG.

[0113] 2E, a small portion of the excitation light may be reflected by the excitation filter to be accordingly sampled by the normalization photodetector. By way of example and not limitation, the normalization photodetector system may be configured to monitor and / or maintain the excitation light at particular reference levels, e.g., by adjusting one or more intensities associated with one or more of the light sources. By way of example and not limitation, one or more intensities may be adjusted to their respective factory calibrated levels over the lifetime of the system. The purpose, working, and integration of a normalization system in the context of a detection system is described in further detail elsewhere in this document.

[0114] In particular embodiments, a first dichroic filter (e.g., DBS1 in FIG.2E) may be provided to receive the multiple bands from the excitation filter. By way of example and not limitation, for n bands of excitation wavelengths, the first dichroic filter may be a 2n-band dichroic filter.

[0115] In particular embodiments, n bands of emission light wavelengths may be generated within the detection well based on illumination by n bands of excitation light wavelengths. In particular embodiments, the emission light directed away from the detection well may be referred to herein as return light, the emission or return light comprising n return signals and / or fluorescent signals. In particular embodiments, the n return signals may be passed from a second or return conduit to a dichroic filter configured pass the n return signals to one or more photodetectors, and to block all other light. In particular embodiments, such as depicted in FIG. 2E by way of example and not limitation, the n return or fluorescent signals may be passed from the light pipe waveguide to the first dichroic filter (DBS1 in FIG. 2E), wherein the first dichroic filter may be accordingly configured to pass only the n return signals for detection of the n return or fluorescent signals by one or more downstream photodetectors. By way of example and not limitation, three downstream photodetectors (PD1, PD2, and PD3) for detecting a plurality of fluorescent signals are depicted in the non-limiting example for FIG. 2E, along with an arrangement of optical modules and components configured to suitably filter and / or direct the return signal to the photodetectors.

[0116] In particular embodiments, the system may be strategically designed for detection of the n return or fluorescent signals while optimizing particular design criteria. By way of example and not limitation, the system may be configured to minimize intra-channel bleed over or cross-talk. By way of example and not limitation, the system may be configured for improving signal-to-noise ratio of detection, e.g., over background noise. While particular methods, devices, and systems described herein are configured to provide such benefits, it will be appreciated that thedisclosure is not limited to the specific arrangements and methods disclosed as non-limiting examples. Other suitable arrangements and variations are possible to provide the disclosed functionality and benefits, and such alternative arrangements and variations are fully contemplated herein.

[0117] In particular embodiments, sequential interrogation of a detection well may be used while providing low cross-talk. In particular embodiments, sequential interrogation may comprise distributing the n bands of excitation light wavelengths into groups that are spectrally distant from each other. In particular embodiments, each group of bands may be simultaneously activated to illuminate the detection well.

[0118] In particular embodiments, particular schemes may be employed to sample the interrogation of the detection well by suitably distributing the n bands of excitation light wavelengths into g groups. By way of example and not limitation, FIG. 2F depicts an exemplary scheme for distributing 8 bands of excitation light wavelengths into 3 groups and employing 8 exemplary dyes in a detection well, according to particular embodiments. FIG. 2G schematically depicts dye absorption and dye emission spectra associated with the 8 dyes provided in a detection well and interrogated according to the exemplary scheme of FIG. 2F. By way of example and not limitation, bands depicted in FIG. 2G using broken lines schematically indicate example excitation filters and emission filters that may be employed in conjunction with the exemplary scheme. It will be appreciated that particular dyes and / or precise location of the filters and / or center wavelengths of bands of excitation light wavelengths may be suitably altered and optimized for reducing cross-talk and / or improving signal-to-noise of detection, and the disclosed examples are non-limiting and provided for illustration only.

[0119] In particular embodiments, a number of groups and / or suitable distribution of multiple bands of excitation light wavelengths into such groups may be determined based on keeping at least 1stnearest spectral neighbors from cross-talking. Separately or additionally, in particular embodiments, a grouping scheme may be determined such that cross-talk associated with both 1stand 2ndnearest spectral neighbors is mitigated. In particular embodiments, higher order cross-talk mitigation may be dispositive for determining a suitable distribution of bands per a grouping scheme.

[0120] An example of the multiplex system that analyzes a plurality of amplified fluorescent signals to analyze multiple analytes, e.g., multiple target nucleic acids, is described in FIGs. 2E, 2H, and 11.For example, the system depicted in FIG. 2E can operate with n emitters / light sources. These emitters can have LED emitters or LED array. The n emitters can provide a number of flash exposures, e.g., 2 to 5 flash exposures (e.g., first flash, second flash, third flash, fourth flash, and / or fifth flash exposures). Each flash exposure provides excitation light having wavelengths or bands of wavelengths in one group. The different flash exposures can be temporally separated, for example, separated by between 0.05 to 0.15 seconds, such as 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 1.14, or 0.15 seconds.

[0121] The light produced by the flash exposures from the one or more emitters are incident through one or more optical components, on a sample in a detection well. In the detection well, one or more fluorescent dyes are excited with the excitation light having specific wavelengths within the two or more flash exposures. Depending on the presence / absence of specific fluorescent dyes and their intensities, the emission fluorescent signals of multiple wavelengths corresponding to the two or more flashes of excitation light are produced and exit the detection well. Different bands of emission fluorescent signals are produced from different bands of excitation light. The emission fluorescent signals can be passed from the light pipe waveguide for further beam splitting and photodetection.

[0122] For example, a first band of emission light generated in response to illumination by the first flash exposure is directed through a suitable array or optical path and delivered to different photodetectors, e.g., a first photodetector and a second photodetector (e.g., PD1 and PD2 of FIGS.

[0123] 2E and 2H). The first photodetector (e.g., PD1) is configured to detect the emission light associated with one wavelength within the first flash exposure and corresponding excitation wavelength of the corresponding fluorescent dye and the second photodetector (e.g., PD2) is configured to detect the emission light associated with another wavelength within the first flash exposure and corresponding excitation wavelength of the corresponding fluorescent dye. In particular embodiments, a suitable first filter (e.g., emission filter I of FIGS. 2E and 2H) may be employed to suitably filter the received emission light prior to receipt by the first photodetector (e.g., PD1) of the fluorescent signal associated the corresponding fluorescent dye. Similarly, a suitable second filter (e.g., emission filter 2 of FIGS. 2E and 2H) may be employed to suitably filter the received emission light prior to receipt by the second photodetector (e.g., PD2) of the fluorescent signal as sociated the corresponding fluorescent dye. Accordingly, the first photodetector and the second photodetector (e.g., PD1 and PD2) are provided with return or fluorescent signals associated with the first flash exposure.A second group of fluorescent dyes may be subsequently activated, for example, via second flash exposure. In particular embodiments, the emission fluorescent signals associated with the excitation light of second group from the second flash exposure having specific wavelengths may be routed to different photodetectors, e.g., a first photodetector and a second photodetector (e.g., PD1 and PD2 of FIGS. 2E and 2H). For example, the first photodetector (e.g., PD1) can be configured to detect the emission light associated with one wavelength within the second flash exposure and corresponding excitation wavelength of the corresponding fluorescent dye and the second photodetector (e.g., PD2) is configured to detect the emission light associated with another wavelength within the second flash exposure and corresponding excitation wavelength of the corresponding fluorescent dye. In particular embodiments, a suitable first filter (e.g., emission filter 1 of FIGS. 2E and 2H) may be employed to suitably filter the received emission light prior to receipt by the first photodetector (e.g., PD1) of the fluorescent signal associated the corresponding fluorescent dye. Similarly, a suitable second filter (e.g., emission filter 2 of FIGS. 2E and 2H) may be employed to suitably filter the received emission light prior to receipt by the second photodetector (e.g., PD2) of the fluorescent signal associated the corresponding fluorescent dye. Accordingly, the first photodetector and the second photodetector (e.g., PD1 and PD2) are provided with emission fluorescent signals associated with the second flash exposure.

[0124] Continuing further, the third group of fluorescent dyes may be activated during a third flash exposure. In particular embodiments, the emission fluorescent signals associated with the third group of excitation wavelengths of light from the third flash exposure may be routed to different photodetectors, e.g., a first photodetector and a third photodetector (e.g., PD1 and PD3 of FIGS. 2E and 2H). For example, the first photodetector (e.g., PD1 ) can be configured to detect the emission light associated with one wavelength within the third flash exposure and corresponding emission wavelength of the corresponding fluorescent dye and the third photodetector (e.g., PD3) is configured to detect the emission light associated with another wavelength within the third flash exposure and corresponding excitation wavelength of the corresponding fluorescent dye. In particular embodiments, a suitable first filter (e.g., emission filter 1 of FIGS. 2E and 2H) may be employed to suitably filter the received emission light prior to receipt by the first photodetector (e.g., PD1) of the fluorescent signal associated the corresponding fluorescent dye. Similarly, a suitable third filter (e.g., emission filter 3 of FIGS.

[0125] 2E and 2H) may be employed to suitably filter the received emission light prior to receipt by the third photodetector (e.g., PD3) of the fluorescent signal associated the corresponding fluorescentdye. Accordingly, the second photodetector and the third photodetector (e.g., PD2 and PD3) are provided with return or fluorescent signals associated with the third flash exposure.

[0126] Continuing further, the fourth group of fluorescent dyes may be activated during a fourth flash exposure. In particular embodiments, the emission fluorescent signals associated with the fourth group of excitation wavelengths of light from the fourth flash exposure may be routed to different photodetectors, e.g., a third photodetector and a fourth photodetector (e.g., PD3 of FIG.

[0127] 2E and PD4 (not shown in FIGS. 2E and 2H). For example, the third photodetector (e.g., PD3) can be configured to detect the emission light associated with one wavelength within the fourth flash exposure and corresponding excitation wavelength of the corresponding fluorescent dye and the fourth photodetector (e.g., PD4) is configured to detect the emission light associated with another wavelength within the fourth flash exposure and corresponding excitation wavelength of the corresponding fluorescent dye. In particular embodiments, a suitable third filter (e.g., emission filter 3 of FIGS. 2E and 2H) may be employed to suitably filter the received emission light prior to receipt by the first photodetector (e.g., PD3) of the fluorescent signal associated the corresponding fluorescent dye. Similarly, a suitable fourth filter (e.g., emission filter 4, not shown in of FIG. 2E) may be employed to suitably filter the received emission light prior to receipt by the fourth photodetector (e.g., PD4) of the fluorescent signal associated the corresponding fluorescent dye. Accordingly, the third photodetector and the fourth photodetector (e.g., PD3 and PD4) are provided with return or fluorescent signals associated with the third flash exposure.

[0128] Similarly, additional groups of fluorescent dyes may be activated during additional flash exposure. The emission fluorescent signals associated with such additional groups of excitation wavelengths of light from the additional flash exposures may be routed to different photodetectors. A person of ordinary skill in the art can design such additional flash exposures and additional photodetectors as well as corresponding optical paths to deliver the excitation signals to different photodetectors. Such embodiments are within the purview of this disclosure.

[0129] Different fluorescent dyes that are activated by the a flash exposure are selected such the fluorescent dyes in one group have the excitation wavelengths that are close to each other on the wavelength spectrum. Thus, different fluorescent dyes in a group can be excited with a light beam of a narrow wavelength band or even with a monochromatic light. Also, different fluorescent dyes that are activated by the a flash exposures are selected such the fluorescent dyes in that group have emission wavelengths that are distinguishable from each other and, hence, can be directed to different photodetectors for detection.For example, as exemplified in connection with FIG. 2H, the fluorescent dyes excited by the first flash exposure are FAM and ATTO 490LS. FAM (5-Carboxyfluorescein) is a fluorescent dye that has the excitation wavelength around 490-495 nm and emits light with a wavelength around 515-520 nm. ATTO490LS is a fluorescent dye that has the excitation wavelength around 490-496 nm and emits light of the wavelength of around 655-661 nm. The excitation signals from FAM are detected in a first photodetector (PD1 of FIG. 2H) and the excitation signals from ATTO490LS are detected in a second photodetector (PD2 of FIG. 2H).

[0130] Similarly, as exemplified in connection with FIG. 2H, the fluorescent dyes excited by the second flash exposure are Cal 610 and Quasar 670. Cal 610 is a fluorescent dye that has the excitation wavelength around 590 nm and emits light with a wavelength around 610 nm. Quasar 670 is a fluorescent dye that has the excitation wavelength around 644-647 nm and emits light of the wavelength of around 666-670 nm. The excitation signals from Cal 610 are detected in a first photodetector (PD1 of FIG. 2H) and the excitation signals from Quasar 670 are detected in a second photodetector (PD2 of FIG. 2H).

[0131] Further, as exemplified in connection with FIG. 2H, the fluorescent dyes excited by the third flash exposure are Cal 560 and ATTO700. Cal 560 is a fluorescent dye that has the excitation wavelength around 538 nm and emits light with a wavelength around 560 nm. ATTQ700 is a fluorescent dye that has the excitation wavelength around 700 nm and emits light of the wavelength of around 716 nm. The excitation signals from Cal 560 are detected in a third photodetector (PD3 of FIG. 2H) and the excitation signals from ATTO700 are detected in a first photodetector (PD1 of FIG. 2H).

[0132] Additional such groups of fluorescent dyes and their corresponding excitation wavelengths or wavelength bands as well as excitation wavelengths or excitation wavelength bands can be readily designed by a skilled artisan and such embodiments are within the purview of the disclosure.

[0133] In some cases, different photodetectors, e.g., PD1 and PD2 of FIGS. 2E and 2H, are configured to detect emission lights of different wavelengths. For example, as shown in FIG. 2H, PD1 is configured to detect three wavelengths associated with FAM, Cal 610, and ATTQ700. While these wavelengths are distinguishable from each other, the detection of these wavelengths is also temporally separated. In other words, the system disclosed herein is configured to determine that: the detection in PD1 of the emission light associated with FAM temporally matches with the timing of the first flash exposure, the detection in PD1 of the emission lightassociated with Cal 610 temporally matches with the timing of the second flash exposure, and the detection in PD1 of the emission light associated with ATTO700 temporally matches with the timing of the third flash exposure.

[0134] Similarly, as shown in FIG. 2H, PD2 is configured to detect two wavelengths associated with ATTO490LS and Quasar 670. While these wavelengths are distinguishable from each other, the detection of these wavelengths is also temporally separated. In other words, the system disclosed herein is configured to determine that: the detection in PD2 of the emission light associated with ATTO490LS temporally matches with the timing of the first flash exposure and the detection in PD2 of the emission light associated with Quasar 670 temporally matches with the timing of the second flash exposure.

[0135] Further, as shown in FIG. 2H, PD3 is configured to detect the wavelengths associated with Cal 560. While PD3 only detects one wavelength, the system disclosed herein is configured to determine that the detection in PD3 of the emission light associated with Cal 560 temporally matches with the timing of the third flash exposure.

[0136] Following the non-limiting example depicted in FIG. 2E and using the non-limiting grouping scheme depicted in FIGs.2F and 2G for continued illustration, an example of operation during an interrogation time interval can be further described as follows. The bands of excitation light wavelengths corresponding to a first group may be first activated. By way of example and not limitation, separate LEDs corresponding to the first group may be enabled, and their respective excitation light may be filtered through a suitable filter (e.g., dichroic filter DBS1) and subsequently provided to the detection well via a suitable conduit. Emission light is generated in response to illumination by the first group of this non-limiting example and directed through a suitable array or optical path and delivered to a photodetector (PD1), wherein PD1 is dedicated to detection of the emission light associated with the first group. In particular embodiments, a suitable filter (e.g., emission filter 1) may be employed to suitably filter the received emission light prior to receipt of the return or fluorescent signal associated with group 1 by photodetector PD1. Accordingly, a photodetector PD1 is provided with return or fluorescent signals corresponding to the three bands of emission light to the photodetector PD1.

[0137] Following the above non-limiting example, a second group may be subsequently activated. In particular embodiments, the return or fluorescent signals associated with the second group of excitation wavelengths of light may be routed to a second photodetector (e.g., PD2 in FIG. 2E).

[0138] In particular embodiments, the emission light associated with the second group may be passed bya second dichroic filter, e.g., DBS2. In particular embodiments, a suitable filter (e.g., emission filter 2) may be employed to suitably filter the received emission light prior to receipt of the return or fluorescent signal associated with group 2 by photodetector PD2. Continuing further with the above non-limiting example, the third group may be finally activated during a final sub-interval of an interrogation time interval. In particular embodiments, the return or fluorescent signals associated with the third group of excitation wavelengths of light may be routed to a third photodetector (e.g., PD3 in FIG. 2E). In particular embodiments, the emission light associated with the third group may be passed by a third dichroic filter, e.g., DBS3. In particular embodiments, a suitable filter (e.g., emission filter 3) may be employed to suitably filter the received emission light prior to receipt of the return or fluorescent signal associated with group 3 by photodetector PD2.

[0139] In particular embodiments, one or more return signals may be time integrated, for e.g., to collect faint charge packets and / or otherwise improve sensitivity. By way of example and not limitation, such time integration may be performed by setting a suitable exposure timescale, such as used in flash photography. By way of example and not limitation, a suitable exposure may be determined based on an energy requirement (in Joules) as the product of the optical energy (in Watts) and the optical integration time (in seconds). In particular embodiments, an integration time may lie between 20 and 120 ms. By way of example and not limitation, a nominal integration time may be 80 ms.

[0140] Further following the above non-limiting example, Group 1 interrogation may integrate three charge packets on three independent charge amplifiers, which are symbolically depicted as triangles in FIG. 2E. By way of example and not limitation, a suitable charge amplifier such as a Texas Instruments IM102 may be employed in particular embodiments. In particular embodiments, the charges may be held and addressed using an analog multiplexer to a single analog-to-digital (ADC) converter to create a digital serial stream of the three channels associated with Group 1. In particular embodiments, after this completes, the second group may be exposed and integrated in the same manner. Finally, continuing with the above non-limiting example, the third group of the final two dyes are exposed and read out, thereby completing the sequence of reading out the 8 digitized return or fluorescent signals. By way of example and not limitation, using the exemplary 80 ms integration and assuming that the integration time dominated the cycle time, the full sequence can be performed in about 0.24 seconds.

[0141] In particular embodiments, such as described in the example of FIG. 2E by way of example, there may be no moving parts in the implementation. By way of example and notlimitation, such an implementation can decrease readout time through simultaneous excitations and / or elimination of filter movements. By way of example and not limitation, such an implementation can increase part reliability, and / or decrease the power and module size. In particular embodiments and as discussed elsewhere herein, aspects of low-cost light pipe collimation (e.g., comprising plastic), and / or optical containment may be beneficially employed.

[0142] As another example, and with reference to FIGs. 2E, 2H, and 11 for purpose of illustration and not limitation, the systems of FIGs. 2E and 2H can operate with 5 sources (from the LED emitter or LED array), 3 flash exposures, 3 photodetectors, and 1 serial data stream. For purpose of illustration and not limitation, FIG. 11 provides an exemplary chart of the percent transmission of DBS1, DBS2, DBS3, the excitation filter (labeled as E0 Filter in FIGs. 2H and 11), EFB1 (labeled as E1 filter in FIGs. 2H and 11), EFB2 (labeled as E2 filter in FIGs. 2H and 11), and EFB3 (labeled as E3 filter in FIGs. 2H and 11), which can be used in accordance with the disclosed subject matter.

[0143] The 5 sources, in the form of 5 LED emitters in an LED array, can emit excitation light bands in the following wavelengths: 1) source 1 (Si): 475 nm; 2) source 2 (S2): 535 nm; 3) source 3 (S3): 590 nm; 4) source 4 (S4): 645 nm; and 5) source 5 (S5): 705 nm. The light can be emitted in a digital serial stream in the following order: S1 . . . S3 . . . S4 . . . S5 . . . S2. The light from the 5 sources can be emitted over about 0.24 seconds, with S1 emitted over a first 0.08 flash (F1); S3 and S4 emitted over a second 0.08 flash (F2); and S5 and S2 emitted over a third 0.08 flash (F3). About 15 seconds can be provided before the pattern of flashes is repeated.

[0144] The five light sources can be directed through an excitation filter. As noted above, a small portion of the excitation light may be reflected by the excitation filter to be accordingly sampled by the normalization photodetector. By way of example and not limitation, the normalization photodetector system may be configured to monitor and / or maintain the excitation light at particular reference levels, e.g., by adjusting one or more intensities associated with one or more of the light sources. By way of example and not limitation, one or more intensities may be adjusted to their respective factory calibrated levels over the lifetime of the system. The excitation filter can be designed for the transmission and blocking as shown in Tables 1 and 2.

[0145] Transmission (T) Wavelength

[0146] T(avg) > 93% 450-480 nm

[0147]

[0148] T(avg) > 93% 526-537 nm

[0149] T(avg) > 93% 584-593 nm

[0150] T(avg) > 93% 640-651 nm

[0151] T(avg) > 93% 700-705 nm

[0152]

[0153] Table 1: Transmission of Excitation Filter

[0154] Optical Density (OD) Wavelength

[0155] OD (avg) > 5 200 - 275 nm

[0156] OD (avg) > 5 275 - 437 nm

[0157] OD (avg) > 5 494-511 nm

[0158] OD (avg) > 5 553 - 568 nm

[0159] OD (avg) > 5 611 - 622 nm

[0160] OD (avg) > 5 670 - 680 nm

[0161] OD (avg) > 5 725 - 850 nm

[0162] OD (abs) > 3.5 488

[0163] OD (abs) > 3.5 518.5 nm

[0164] OD (abs) > 3.5 545 nm

[0165] OD (abs) > 3.5 475 nm

[0166] OD (abs) ≥ 3.5 600 nm

[0167] OD (abs) ≥ 3.5 631 nm

[0168] OD (abs) ≥ 3.5 660.5 nm

[0169] OD (abs) ≥ 3.5 690 nm

[0170]

[0171] OD (abs) > 3.5 715 nm

[0172]

[0173] Table 2: Blocking of Excitation Filter

[0174] Excitation light that passes through the excitation filter can be reflected by DBS1. DBS1 can be designed for transmission and reflection as shown in Tables 3 and 4.

[0175] Transmission (T) Wavelength

[0176] T(avg) > 93% 494 - 511 nm

[0177] T(avg) > 93% 553 - 568 nm

[0178] T(avg) > 93% 611 - 622 nm

[0179] T(avg) > 93% 670 - 680 nm

[0180] T(avg) > 93% 725 - 850 nm

[0181]

[0182] Table 3: Transmission of DBS1

[0183] Reflection (R) Wavelength

[0184] R(avg) > 98% 450 - 480 nm

[0185] R(avg) > 98% 526 - 537 nm

[0186] R(avg) > 98% 584 - 593 nm

[0187] R(avg) > 98% 640 - 651 nm

[0188] R(avg) > 98% 700 - 705 nm

[0189]

[0190] Table 4: Reflection of DBS1

[0191] Based on the design of DBS1, as shown above, the excitation light from each of the five excitation sources (S1-S5) can be reflected by DBS1, directed through the waveguide, and into the detection wells. Light from the excitation sources can impinge dyes and result in 6 emission bands of emission light. For example, but not limitation, light from S1 can impinge on FAM dye to produce a first emission light λ1. Likewise, light from S1 can also impinge on ATTO490LS dye to produce a second emission light λ2. Light from S2 can impinge on Cal 560 dye to produce a third emissionlight λ3. Light from S4 can imping on Cal 610 dye to produce a fourth emission light λ4. Light from S5 can impinge on Quasar 670 dye to produce a fifth emission light λ5. Light from S6 can impinge on ATTO700 dye to produce a sixth emission light λ6. Emission light λ1-λ6 can be directed back through the waveguide to DBS1, with each of λ1-λ6 passing through DBS1.

[0192] After passing through DBS1, λ1-λ6 can impinge DBS3. DBS3 can be designed for transmission and reflection as shown in Tables 5 and 6.

[0193] Transmission (T) Wavelength

[0194] T(avg) > 93% 553 - 568 nm

[0195]

[0196] Table 5: Transmission of DBS3

[0197] Reflection (R) Wavelength

[0198] R(avg) > 98% 450 - 537 nm

[0199] R(avg) > 98% 584 - 850 nm

[0200]

[0201] Table 6: Reflection of DBS3

[0202] Based on the design of DBS3, as shown above, λ1-λ2 and λ4-λ6 can be reflected by DBS3, and λ3 can be transmitted through DBS3. Reflected λ1-λ2 and λ4-λ6 can impinge on DBS2, which can be designed for transmission and reflection as shown in Tables 7 and 8.

[0203] Transmission (T) Wavelength

[0204] T(avg) > 93% 670 - 680 nm

[0205]

[0206] Table 7: Transmission of DBS2

[0207] Reflection (R) Wavelength

[0208] R(avg) > 98% 450 - 651 nm

[0209] R(avg) > 98% 570 - 850 nm

[0210]

[0211] Table 8: Reflection of DBS2Based on the design of DBS2, as shown above, λ1, λ4, and λ6 can be reflected by DBS2, and λ2 and λ5 can be transmitted through DBS2.

[0212] After being reflected by DBS2, emission bands Xi, X4, and X& can impinge emission filter bands 1 (EFBi), which can be designed for transmission and blocking as shown in Tables 9 and 10.

[0213] Transmission (T) Wavelength

[0214] T(avg) > 93% 494 - 511 nm

[0215] T(avg) > 93% 611 - 622 nm

[0216] T(avg) > 93% 725 - 850 nm

[0217]

[0218] Table 9: Transmission of EFB1

[0219] Optical Density (OD) Wavelength

[0220] OD (avg) > 5 200 - 275 nm

[0221] OD (avg) > 6 275 - 480 nm

[0222] OD (avg) > 6 450 - 480 nm

[0223] OD (abs) ≥ 3.5 488 nm

[0224] OD (abs) ≥ 3.5 518.5 nm

[0225] OD (avg) > 6 526 - 593 nm

[0226] OD (avg) > 6 526 - 537 nm

[0227] OD (avg) > 6 584 - 593 nm

[0228] OD (abs) ≥ 3.5 600 nm

[0229] OD (abs) ≥ 3.5 631 nm

[0230] OD (avg) > 6 640 - 705 nm

[0231]

[0232] OD (avg) > 6 640 - 651 nm

[0233] OD (avg) > 6 700 - 705

[0234] OD (abs) > 3.5 715 nm

[0235]

[0236] Table 10: Blocking of EFB1

[0237] Based on the design of EFB1, as shown above, λ1, λ4, and λ6 can pass through EFB1 and impinge on PD1, where the values can be measured as noted above. If necessary, a mirror can be used between DBS2 and EFB1 to redirect light as necessary. Likewise, additional mirrors can be provided along other paths as necessary to redirect light.

[0238] After being transmitted through DBS2, λ2 and λ5 can impinge EFB2, which can be designed for transmission and blocking as shown in Tables 11 and 12.

[0239] Transmission (T) Wavelength

[0240] T(avg) > 93% 670 - 680 nm

[0241]

[0242] Table 11: Transmission of EFB1

[0243] Optical Density (OD) Wavelength

[0244] OD (avg) > 5 200 - 275 nm

[0245] OD (avg) > 6 275 - 651 nm

[0246] OD (avg) > 6 450 - 480 nm

[0247] OD (avg) > 6 526 - 537 nm

[0248] OD (avg) > 6 584 - 593 nm

[0249] OD (avg) > 6 640 - 651 nm

[0250] OD (abs) ≥ 3.5 660.5 nm

[0251] OD (abs) ≥ 3.5 690 nm

[0252] OD (avg) > 6 700 - 705 nm

[0253]

[0254] OD (avg) > 6 700 - 850 nm

[0255]

[0256] Table 12: Blocking of EFB2

[0257] Based on the design of EFB2, as shown above, λ2 and λ5 can pass through EFB2 and impinge on PD2, where the values can be measured as noted above.

[0258] After being transmitted through DBS3, λ3 can impinge EFB3, which can be designed for transmission and blocking as shown in Tables 13 and 14.

[0259] Transmission (T) Wavelength

[0260] T(avg) > 93% 553 - 568 nm

[0261]

[0262] Table 13: Transmission of EFB3

[0263] Optical Density (OD) Wavelength

[0264] OD (avg) > 5 200 - 275 nm

[0265] OD (avg) > 6 275 - 537 nm

[0266] OD (avg) > 6 450 - 480 nm

[0267] OD (avg) > 6 526 - 537 nm

[0268] OD (abs) ≥ 3.5 545 nm

[0269] OD (abs) ≥ 3.5 576nm

[0270] OD (avg) > 6 584 - 593 nm

[0271] OD (avg) > 6 584 - 850 nm

[0272] OD (avg) > 6 640 -651 nm

[0273] OD (avg) > 6 700 - 705 nm

[0274]

[0275] Table 15: Blocking of EFB3

[0276] Based on the design of EFB3, as shown above, λ3 can pass through EFB3 and impinge on PD3, where the values can be measured as noted above.A summary of the configuration described above is provide below in Table 15. The configuration can be implemented using wrap around electronics.

[0277] Excitation Excitation Flash Dye Z Photodetector

[0278] Source wavelength

[0279] S1 475 nm F1 FAM λ1 PD1

[0280] S1 475 nm F1 ATTO490LS λ2 PD2

[0281] s2535 nm F3Cal 560 PD3

[0282] S3 590 nm F2Cal 610 X4 PDi

[0283] s4645 nm F2Quasar 670 Z5 PD2

[0284] S5 705 nm F3 ATTO700 λ6 PD1

[0285]

[0286] Table 16: Summary of configuration

[0287] In place of the dyes or combination of the dyes described above, certain additional dyes can also be used in the devices and methods disclosed herein. For example, Chromeo494 dye can be used in place of ATTO490LS.

[0288] In some cases, additional channels, for example, emitters that can emit excitation light bands in those described above for S1 to S5 or emitters that can emit excitation light in additional or different wavelength bands. In certain such cases, additional dyes such as DY 52 IXL or DY 485XL can be used. DY-521XL dye is a fluorophore known for its large stokes shift, exciting around 533 nm and emitting around 674 nm (far-red). DY 485XL is a fluorophore having an excitation peak at around 485 nm and an emission peak at around 560 nm.

[0289] FIG. 3B represents a schematic of a system for detecting fluorescence, according to particular embodiments of the present disclosure. The second schematic of the system includes a plurality of light sources arranged on a single plane in a circular configuration. Each light source in the plurality of light sources is configured for emitting light at a specified excitation wavelength in a sequential manner to illuminate a specified detection well of the plurality of detection wells. The light sources are light emitting diodes (LEDs) arranged with a collimating lens ring. A dichroic beam splitter is shown and is arranged at an angle ranging from 30-60 degrees with reference to the single plane. The dichroic beam splitter is configured for reflecting the specifiedexcitation wavelength to the conduit and for transmitting an emission wavelength received from the conduit to the photodetector. An excitation filter is shown to be arranged on the same plane and is configured for transmitting the specified excitation wavelength towards the dichroic beam splitter. An emission focus lens and a parabolic focus mirror is shown. A photodetector is shown and a conduit for transmitting an emission wavelength is shown. The photodetector is on the same single plane as the plurality of light sources. The conduit is in the form of light pipes which enable transmission of emission wavelength emitted from the plurality of detection wells to the photodetector. An emission filter is shown to be arranged on the same plane and is configured for transmitting the specified emission wavelength towards the photodetector.

[0290] FIG. 9A represents an exemplary system for detecting fluorescence, according to particular embodiments of the present disclosure. This system includes a plurality of light sources arranged on a single plane, e.g., a single printed circuit board (PCB). This system includes a light source (m) and another light source (m+1). Each light source in the plurality of light sources is configured for emitting light comprising specified excitation wavelengths to illuminate a specified detection well of the plurality of detection wells. For example, the system includes a detection well (n) and another detection well (n+1). Detection well n is associated with light source m such that light emitted by m is directed to n. The system may include one or more photodetectors. For example, the system includes a photodetector (h) and another photodetector (h+1). An excitation filter is configured for transmitting the excitation wavelengths. An excitation filter is positioned in an optical path between the light source and a photodetector. An emission filter is positioned in an optical path between the detection well and the photodetector. Another excitation filter is disposed in an optical path between the detection wells and the photodetectors. This excitation filter collimates the excitation light exited from detection wells and directs it to a normalization photodetector. The normalization photodetector determines whether the exited excitation light from different light sources is substantially similar in intensity. The system may include plurality of excitation filters each in an optical path between a detection well and a normalization photodetector. The system may include a single excitation filter that steers light from different light sources to a single normalization photodetector. The system may include plurality of excitation filters that steer excitation light to a normalization photodetector. The normalization detector is also shown which compares the intensity of excitation light emitted by the light sources. If the intensity of the exited excitation light detected by the normalization detector is not substantially identical, then the normalization detector indicates to the processor that normalization of excitation light sources needs to be performed. The processor carries normalization process by adjusting the intensity of light emitted by the light source. The systemmay have a single normalization detector and at least one photodetector. In some cases, the system may have a single photodetector which may be used for both normalization and fluorescence detection.

[0291] Excitation light may enter the detection wells and a majority of the excitation light exits from the detection wells and is directed to the normalization photodetector. Emitted light (fluorescent light) from the wells is directed to a photodetector. An emission filter transmits light emitted from the excited fluorophore to the photodetector. Another filter (e.g., the excitation filter) transmits the excitation light to the normalization photodetector. This can be performed by a dichroic filter which steers emission light and excitation light in two directions to either photodetector.

[0292] As would be understood, the physical location of the filters in relation to a light source and a photodetector can be varied by using light conduits that determine the optical path of light. A light conduit may direct light from a light source to the detection wells, where the light source and detection wells are located on different planes. A light conduit may direct light from a light source to a light filter. A light conduit may direct light from a light filter to a detection well. A light conduit may direct light from a detection well to a light filter. A light conduit may direct light from a light filter to a photodetector. The use of light conduits can add flexibility in physical arrangement of the individual components of the systems disclosed herein.

[0293] FIG. 9B represents a schematic of a system for detecting fluorescence, according to particular embodiments of the present disclosure. This system includes a plurality of light sources arranged on a single plane. For example, the system includes a light source (n) and another light source (n+1). Each light source in the plurality of light sources is configured for emitting excitation light comprising specified excitation wavelengths to illuminate a specified detection well of the plurality of detection wells. A light source for illuminating a detection well for exciting a fluorophore is also referred to herein as an excitation source. In FIG. 9B, a light source (n(λ1)) emits light at excitation wavelengths λ1 and a light source (n(λ2)) emits light at excitation wavelengths λ2 in sequential manner to illuminate detection well (n). Similarly, light source (n+1(λ1)) emits light at excitation wavelengths (λ1) and a light source (n+1(λ2)) emits light at excitation wavelengths (λ2) in sequential manner to illuminate detection well (n+1). In an example, the sequence in which the light sources are turned on to emit excitation light may be n(λ1), followed by n(λ2), followed by n+1(λ1), and finally n+1(λ2). The system includes a detection well (n) and another detection well (n+1). The system may include one or more photodetectors. For example, the system includes a photodetector (n) and another photodetector(n+1). In some examples, the system may include a single photodetector for detecting light emitted from multiple detection wells. An excitation filter is shown to be arranged adjacent to the light source and is configured for transmitting specified excitation wavelengths to a detection well. An emission filter is positioned adjacent the detection well. The emission filter can transmit light having wavelengths comprising the wavelengths of the fluorescent signal and block other wavelengths. A light conduit (e.g., light pipes) may transmit the emission light, exiting the emission filter, to a photodetector (also referred to as a fluorescent detector). Another excitation filter is disposed adjacent the detection wells. This excitation filter collimates the excitation light exited from detection wells and directs it (e.g., through a light conduit) to a normalization detector. The system may include plurality of excitation filters between the detection wells and the photodetectors. The normalization detector compares the intensity of excitation light emitted by the light sources. If the intensity of the excitation light from different light sources detected by the normalization detector is not substantially identical, then the normalization detector indicates to the processor that normalization of excitation light sources needs to be performed. The processor carries normalization process by compensating the intensity. The system may have a normalization detector and a photodetector. In some cases, the system may have a single detector which may be used for both normalization and fluorescence detection.

[0294] FIG. 4A shows configuration of a plurality of light sources, according to particular embodiments. The plurality of light sources are arranged in a concentric configuration. The outer circle of the concentric configuration comprises at least 12 LEDs and the inner circle of the concentric configuration comprises at least 4 LEDs. The LEDs may be turned on in any suitable sequence. An exemplary sequence is shown in FIG. 4A where the number for each LED represented by a circle indicates the sequence in which the LED is turned on.

[0295] FIG.4B shows a schematic of the LEDs shown in the first panel in FIG.4A, according to particular embodiments. A plurality of wavelengths emitted from the LED (10) is shown. The LED (10) is associated with a collimator (15) that changes the angle of the emitted light such that the wavelengths are parallel to each other. The parallel wavelengths are directed to an excitation filter (20) which is also depicted.

[0296] In some embodiments, the light sources are any light source capable of emitting light having a desired range of wavelengths such as light within the visible or ultraviolet spectrum. In some embodiments, the light sources are light emitting diodes (LEDs) or a mercury lamp or laser. In some embodiments, the LEDs are lamp type (leaded). In some embodiments, the LEDs are a chip type (surface mount). In some embodiments, the LEDs are selected based on the type offluorescence molecule present in a sample in the detection wells. The LEDs can generate visible light between about 400 nm and about 750 nm in wavelength. The LEDs could include red, green, blue, and white LEDs, i.e., LEDs that generate red, green, or blue light. The LEDs could also generate ultraviolet light between 100 nm and about 400 nm in wavelength. The LEDs could also generate near infrared light between 800 nm and about 2500 nm in wavelength.

[0297] An advantage of the presently disclosed devices, systems and methods is that they can use generic LEDs, reducing costs. Many devices, systems, and methods for detecting fluorescence use expensive light sources that emit light having tightly defined wavelengths and consistent intensity. Light sources that emit tightly defined wavelengths are quite expensive. Light sources that consistently emit light of the same intensity are quite expensive and have short life. In contrast, the devices and systems disclosed herein include components that can normalize the light emitted from the plurality of light sources allowing use of cheaper light sources. In one example, excitation light from a light source is measured by a photodetector. The photodetector compares the measured light to the excitation light emitted from other light sources in the device. When a difference in intensity of the light emitted by the different light sources is detected, the system, using a processor normalizes the emission light measured by the photodetector by compensating for the difference in the intensity of light emission light. For example, if a 1st light source (e.g., associated with a 1stdetection well) emits light of intensity X, a 2ndlight source (e.g., associated with a 2nd detection well) emits light of intensity 2X, a 3rd light source (e.g., associated with a 3rd detection well) emits light of intensity 0.5X, and so on, the detector can relay the difference to the processor. If the three wells have the same level of fluorescence, due to differences in excitation light intensities, the emitted light would vary in intensity. The processor when relaying emission light detected from the wells can adjust the intensity of the detected emission light such that the light measured as emitted from the 2nd well and 3rd well are normalized to an intensity that compensates for the difference in intensity of the excitation light. Examples of a system that includes a normalization detector are depicted in FIGs. 9A and 9B.

[0298] The presently disclosed devices, systems, and methods control the wavelengths of light entering a detection well by including light filters. The light filters narrow the wavelengths reaching the detection wells relative to the wavelengths emitted by the light sources. Thus, the light sources do not have to be configured for emitting wavelengths in a narrow range, decreasing the cost of the light sources.

[0299] In some embodiments, the LEDs emit an excitation light that is absorbed by fluorescence molecules. In some embodiments, the light source are selected based on the excitationwavelengths for the fluorescent molecule, e.g., an LED that emits light at wavelength of around 460 nm may be selected for dyes, such as, FAM; and an LED that emits light at wavelength of around 570 nm may be selected for dyes, such as, ROX; an LED that emits light at wavelength of around 345 nm may be selected for dyes, such as, Alexa flour; an LED that emits light at wavelength of around 550 nm may be selected for dyes, such as, Cy3; an LED that emits light at wavelength of around 495 nm may be selected for dyes, such as, Fluorescein F1TC; an LED that emits light at wavelength of around 650 nm may be selected for dyes, such as, Cy5; or an LED that emits light at wavelength of around 743 nm may be selected for dyes, such as, Cy7.

[0300] In some embodiments, mthlight source emits light at one excitation wavelength to illuminate nth detection well. In some embodiments, mthlight source emits light at another excitation wavelength to illuminate nth detection well.

[0301] In some embodiments, (m+l)01light source emits light at one excitation wavelength to illuminate (n+1)’11detection well. In some embodiments, (m+1)111light source emits light at another excitation wavelength to illuminate (n+l)thdetection well.

[0302] In some embodiments, a single light source is configured for emitting different excitation wavelengths. For example, a single light source may be configured for emitting 1, 2, 3, 4, or 5 different excitation wavelengths.

[0303] The detection wells may be present in a cartridge, an assay plate, a microfluidic chip and the like. In certain embodiments, the detection wells are made of elastomers or polymers that do not substantially fluoresce. In certain embodiments, the detection wells are made of elastomers or polymers such as PDMS, PMMA, Polyurethane, PFPE, SIFEL®, parylene, and others. In some embodiments, the detection wells are preset in a cartridge, an assay plate, a microfluidic chip, etc., adapted to perform DNA hybridization assays, expression analysis of mRNA, nucleic acid amplification, sandwich ELISA, etc.

[0304] The detection wells may have any suitable shape and dimensions provided they hold sufficient amount of sample. The detection wells may be made from a substantially optically transparent material, e.g., glass or clear plastic, and the like.

[0305] In some cases, the systems and devices described herein are portable. In some cases, the systems and devices described herein have a relatively small size, e.g., size of desktop, laptop, tablet, or smartphone.In some cases, the light conduits, such as, light pipes may have any suitable dimensions and shape. In certain embodiments, the light pipes may have a tapered configuration where the internal space for light transmission may decrease from one end to the other. In certain embodiments, the light pipes may have a larger opening at the end located adjacent to the detection wells and a smaller opening at the end located adjacent to the photodetector.

[0306] Aspects of the present disclosure include a device for detecting the presence of a fluorescent signal in a plurality of detection wells in a cartridge. More specifically, devices of interest comprise a cartridge staging region, and the systems of the present disclosure.

[0307] Devices of interest comprise systems of the present disclosure which are described in the above section.

[0308] In some embodiments, the device of present disclosure includes a cartridge staging region configured to accommodate and secure in position a cartridge comprising the plurality of detection wells. In some embodiments, the device of present disclosure further comprises a cartridge positioned in the cartridge staging region.

[0309] In some embodiments, the device of present disclosure further comprises a sample present in at least some of the plurality of detection wells. In some embodiments, the device of present disclosure is configured for dispensing into the detection wells the sample present in at least some of the plurality of detection wells. In some embodiments, the device of present disclosure configured for preparing the sample, for example, the device isolates an analyte of interest and dispenses the analyte into the plurality of detection wells. In some embodiments, the analyte of interest is fluorescently labeled in the detection wells prior to detection by the device. In some embodiments, the analyte of interest is amplified the detection wells and the amplification product is fluorescently labeled in the detection wells prior to detection by the device.

[0310] In some embodiments, the sample comprises a positive control comprising a first fluorescent component and, if present, a second fluorescent component indicative of presence of an analyte in the sample, wherein the first and second fluorescent components have distinct fluorescence profiles.

[0311] Aspect of the present disclosure includes an optical detection module, comprising: a chamber; an illumination printed circuit board having a plurality of light sources arranged on a single plane in a circular configuration; an illumination optical port on one side of the chamber; a dichroic beam splitter placed in the chamber at an angle ranging from 30-60 degrees with referenceto the illumination optical port; a plurality of light conduits each configured to interrogate one of a plurality of detection wells; or a dynamic steerable mirror configured to interrogate one of a plurality of detection wells; and a light pipe optical port on another side of the chamber configured for unique optical paths; a detection optical port on another side of the chamber; a detection printed circuit board having one or more photodetectors arranged perpendicular to the plane; wherein the plurality of light sources in the illumination printed circuit board are configured for emitting an excitation light at a specified excitation wavelength in a sequential manner such that one detection well is interrogated at a time; the illumination optical port on one side of the chamber is configured for passing a specified excitation wavelength; the dichroic beam splitter is configured for reflecting the specified excitation wavelength to one of the plurality of detection wells and for transmitting an emission wavelength received from the detection well to one of the photodetectors; the detection optical port on another side of the chamber is configured for passing emission wavelength; the photodetector on the detection printed circuit board is configured for measuring the fluorescence from a sample; wherein the chamber, the illumination printed circuit board and the detection printed circuit board are connected by a plurality of cables.

[0312] Another aspect of the present disclosure includes an optical detection module, comprising a chamber; an illumination printed circuit board having a plurality of light sources arranged on a single plane in a circular configuration; an illumination optical port on one side of the chamber; a dichroic beam splitter placed in the chamber at an angle ranging from 30-60 degrees with reference to the illumination optical port; a plurality of light conduits each configured to interrogate one of a plurality of predetermined detection wells; or a dynamic steerable mirror configured to interrogate one of a plurality of predetermined detection wells; and a light pipe optical port on another side of the chamber configured for unique optical paths; a detection optical port on another side of the chamber; a detection printed circuit board having a photodetector arranged perpendicular to the plane; wherein the plurality of light sources in the illumination printed circuit board are configured for emitting an excitation light at a specified excitation wavelength in a sequential manner such that one predetermined detection well is interrogated at a time; the illumination optical port on one side of the chamber is configured for passing a specified excitation wavelength; the dichroic beam splitter is configured for reflecting the specified excitation wavelength to one of the plurality of predetermined detection wells and for transmitting an emission wavelength received from the predetermined detection well to the photodetector; the detection optical port on another side of the chamber is configured for passing emission wavelength; the photodetector on the detection printed circuit board is configured for measuringthe fluorescence from a sample: wherein the chamber, the illumination printed circuit board and the detection printed circuit board are connected by a plurality of cables.

[0313] The photodetector may be any photodetector suitable for detecting emission wavelengths. In certain examples, the photodetector may be a photodiode, e.g., a single photodiode. The photodiode may operate in photovoltaic mode and generate voltage and electric current in a material upon exposure to light. The photodiode may operate in photoconductive mode in which a material becomes more electrically conductive due to the absorption of electromagnetic radiation such as visible light, ultraviolet light, infrared light, or gamma radiation.

[0314] FIG. 6 shows optical detection module as per one of the embodiments of the present disclosure, according to particular embodiments.

[0315] The present disclosure includes a semi-automated or automated method of detecting fluorescence in a plurality of detection wells in a cartridge in the device of the present disclosure, comprising steps of: (a) generating an excitation light from a mthlight source of the plurality of light sources at a specified excitation wavelength and illuminating a nth detection well of the plurality of detection wells and ceasing generating the excitation light; (b) transmitting the emission wavelength emitted from the nth detection well to the photodetector; (c) detecting the emission wavelength on the photodetector; (d) correlating the emission wavelength detected by photodetector to the nth detection well emitting the emission wavelength, and (e) repeating steps (a) to (d) a plurality of times to detect fluorescence in the plurality of detection wells.

[0316] The plurality of light sources may comprise “m” light sources, where each light source is capable of illuminating “n” well of the plurality of detection wells, wherein light source emits an excitation light at different wavelengths and the method comprises generating an excitation light from a mthlight source and illuminating the nth detection well of the plurality of detection wells and ceasing generating the excitation light and conducting steps (b) to (d) and generating an excitation light from (m+1)thlight source and illuminating the (n+1)thdetection well of the plurality of detection wells and ceasing generating the excitation light and conducting steps (b) to (d).

[0317] In certain aspects, the semi-automated or automated method comprises steps of: (a) generating an excitation light with a plurality of light sources at a specified excitation wavelength in a sequential manner such that one of a plurality of predetermined detection wells is illuminated at a time; (b) directing the specified excitation wavelength to a dichroic beam splitter such that it is reflected to a conduit / system to interrogate one of the plurality of predetermined detectionwells; (c) illuminating a sample in one of the plurality of the predetermined detection wells with the specified excitation wavelength to generate an emission wavelength; (d) directing the emission wavelength received from the predetermined detection well to the dichroic beam splitter such that it is transmitted to a photodetector; (e) detecting the emission wavelength in the photodetector; (f) correlating the emission wavelength detected by photodetector to the detection well emitting the emission wavelength.

[0318] Aspects of the present disclosure include semi-automated or automated methods of detecting fluorescence in a plurality of detection wells in a cartridge. In some embodiments, methods of interest comprise (a) generating an excitation light from a first light source of the plurality of light sources at a specified excitation wavelength and illuminating a first detection well of the plurality of detection wells and ceasing generating the excitation light; (b) transmitting the emission wavelength emitted from the first detection well to the photodetector; (c) detecting the emission wavelength on the photodetector; (d) correlating the emission wavelength detected by photodetector to the first detection well emitting the emission wavelength. The method may further comprise (el) repeating steps (a) to (d) a plurality of times to detect fluorescence in the plurality of detection wells; or (e2) wherein the first light source generates a first excitation light in step (a) and the method comprises a repeating steps (a) to (d) wherein first light source generates a second excitation light.

[0319] In some embodiments, the plurality of light sources comprises a pair of light sources, where each pair is capable of illuminating a single well of the plurality of detection wells, wherein each member of the pair emits an excitation light at different wavelengths and the method comprises generating an excitation light from a first member of the first pair of light sources and illuminating the first detection well of the plurality of detection wells and ceasing generating the excitation light and conducting steps (b) to (d) and generating an excitation light from a second member of the first pair of light sources and illuminating the first detection well of the plurality of detection wells and ceasing generating the excitation light and conducting steps (b) to (d).

[0320] In some embodiments, the correlating comprises information regarding the light source used for generating the excitation light.

[0321] In some embodiments, the conduit comprises a plurality of light conduits, wherein there is a 1: 1 correlation between each light conduit and each detection well and the correlating comprises information regarding the light conduit transmitting the emission wavelength to the photodetector.System, Device and Method for Detecting Fluorescent Signal During Amplification of a Target Nucleic Acid

[0322] The systems, devices, and methods described herein can be used for monitoring fluorescent signal during nucleic acid amplification as noted in the preceding sections. The nucleic acid amplification may be carried out in individual detection wells in a cartridge. The cartridge may be as described herein. For example, the cartridge may include the detection wells arranged in a row. The cartridge may include detection wells that are of any shape suitable for accommodating the wells in the cartridge. The cartridge may have any shape suitable for accommodating the cartridge in a device for detecting fluorescence.

[0323] The cartridge can include at least 4 detection wells, at least 5 detection wells, at least 6 detection wells, at least 7 detection wells, or more detection wells. The plurality of light sources may be as described in the preceding sections. At least one light source may be configured for emitting an excitation light to illuminate a single detection well, wherein the excitation light has a specified wavelength. For example, each detection well may be associated with a single light source.

[0324] In another example, each detection well may be associated with multiple light sources that emit light of different wavelengths. In another example, each detection well may be associated with a single light source that emits light having a broad wavelength band and the system includes multiple filters for controlling the wavelengths reaching the detection wells.

[0325] Each of the detection wells may be interrogated through optical excitation and instantaneous fluorescent signal readout. This interrogation may proceed sequentially, e.g., first illuminating a first detection well sequentially with different wavelengths followed by illuminating a second detection well sequentially with the different wavelengths and so on; and repeats periodically.

[0326] The duration of illumination of a detection well with a particular excitation wavelength can be determined empirically based on, e.g., intensity of the excitation wavelength, number of fluorophores in the detection wells, time period of primer extension phase in a PCR cycle, etc.

[0327] The timing of illumination of detection wells in which PCR is being performed may be determined in conjunction with PCR cycle. For example, the detection wells may be illuminated to detect fluorescent signal during the extension phase of the PCR cycle. More particularly, the detection wells may be illuminated to detect fluorescent signal during the end of the extensionphase of the PCR cycle. This interrogation may be repeated periodically in the extension phase during the PCR. This interrogation may be repeated for every extension phase during the PCR. This interrogation may be repeated for every other extension phase during the PCR. This interrogation may be repeated after every 3, every 5, or every 10 extension phases during the PCR.

[0328] As is well known in the field, during PCR cycle, the temperature of a sample containing a target nucleic acid is raised to allow denaturation of the target nucleic acid, followed by lowering of the temperature to allow primers and probe to anneal to the target nucleic acid. Then the temperature is maintained (e.g., at 55°C) to allow for elongation of the primers. The elongation phase may be as short as about 10-5 seconds, e.g., about 8 seconds. When the interrogation of the detection well is to be performed during the elongation phase, the system is programmed to manage this interrogation sequence such that it is synchronized with the elongation phase by obtaining information from a PCR module that controls the heating cycle. The interrogation of a detection well during the elongation phase, may involve illumination with a single type of excitation light to detect, e.g., a single type of fluorescent signal. The interrogation of a detection well during the elongation phase, may involve illumination with multiple types of excitation lights to detect, e.g., multiple types of fluorescent signal.

[0329] FIG. 10 depicts an exemplary detection system for interrogating one or more detection wells during the end of the elongation phase of the PCR cycle, according to particular embodiments. The depicted PCR cycle includes 40 rounds of denaturation and annealing of the primers and probes “annealing” followed by extension of the primers during the elongation phase “extension”. Each detection well is illuminated sequentially by excitation wavelengths 1 through n. n may be 2, 3, 4, or more. Each of the different excitation wavelengths may be configured for detection of a different fluorophore present in the detection well. The different excitation wavelengths may be emitted by different light sources, by a single light source in conjunction with multiple filters, or different light sources in conjunction with a single light filter to direct different wavelengths to the detection wells.

[0330] In the exemplary, non-limiting system depicted in FIG. 10, there are four different wavelength bands (referred to as channels, channel 1 through channel n, where n=4). The system may have 2 detection wells, 3, or 4 detection wells and each well may be associated with a light source that can transit (e.g., through use of appropriate filters that serve as channels) four different wavelength bands to each well. On the optical side of the system, a first color is used to illuminate the first well via channel 1, a second color is used to illuminate the first well via channel 2, a third color is used to illuminate the first well via channel 3, and a fourth color is used to illuminate thefirst well via channel 4. The same procedure is repeated for the second detection well, the third detection well and so on. The optical side of the system coordinates with thermal side of the system which controls the PCR cycle to synchronize the illumination of the wells with the extension phase. In this example, the four light sources that each emit different excitation lights may be associated with a single light filter that is configured to narrow the wavelength band emitted by a light source. Use of the light filter allows low-cost light sources to be used.

[0331] The system is configured to sample each detection well with each of the set of channels at the end of each extension phase in a sequential fashion. In one example, each well may be illuminated for 80 milliseconds per channel. For 40 thermal cycles each channel is sampled 40 times

[0332] For each extension phase, each channel is sampled once. In the case of a system with 4 wells with 4 colors (from 4 different fluorophores) in each well, 16 emission signals are acquired for each extension phase. Based on a sampling duration of 80 milliseconds, the time to sample 16 channels would be about 1.28 seconds. The sequence would involve: WelllColorl, WelllColor2, Weill Color3, Weill Color4, Well2Colorl, Well2Color2, Well2Color3, Well2Color4, Well3Colorl, Well3Color2, Well3Color3, Well3Color4, Well4Colorl, Well4Color2, Well4Color3, Well4Color4. The optical side of the system then pauses activating the light source until another extension phase is nearly complete before activating the light sources and illuminating each of the wells via the four different channels per wells. Each light source may be configured with four sets of channels such that the system includes 16 channels (4 of 4 sets of channels). A single PCR cycle length may be about 10 seconds with 5 seconds annealing and 5 seconds in extension. Interrogation of the detection wells can be performed just prior to start of the annealing phase or about 1 second, about 2 seconds, about 3-4 seconds into the extension phase.

[0333] A normalization detector may sample the excitation lights from different light sources in the system at the beginning of the PCR program. If it is determined that the light sources are emitting excitation light at different intensity, the processor may boost the intensity of light emitted at a lower intensity or lower the intensity of light emitted at a higher intensity to ensure that the different light sources emit light of comparable intensity. Additionally, or alternatively, a normalization detector may sample the excitation lights from different light sources in the system at the beginning, middle, or towards the end PCR program. If it is determined that the light sources are emitting excitation light at different intensities, the processor may adjust the intensity of the measured fluorescence to compensate for difference in intensity of the excitation light.Further provided herein are analyzers that use a cartridge and a system disclosed herein to analyze a sample for target analytes, particularly, multiple target analytes, such as multiple target nucleic acids. Thus, certain aspects of the disclosure provide an analyzer for analyzing one or more target analytes in a sample using an analysis cartridge in a system disclosed herein. In some cases, the analyzer comprises:

[0334] one or more of light sources operably linked to one or more detection wells in an analysis cartridge, wherein each light source in the one or more light sources is configured for emitting an excitation light comprising specified excitation wavelengths in a sequential manner to illuminate a specified detection well in the analysis cartridge;

[0335] one or more photodetectors;

[0336] a conduit for transmitting an emission light comprising emission wavelengths emitted from the one or more detection wells in the analysis cartridge to one of the one or more photodetectors;

[0337] a processor, and

[0338] a non-transitory computer readable medium comprising instructions, which when executed by the processor, cause the analyzer to:

[0339] generate, by one or more light sources, excitation light comprising one or more wavelengths or one or more bands of excitation wavelengths, wherein the one or more wavelengths or one or more bands of excitation wavelengths are distributed into a plurality of groups such that each group is associated with one or more of the one or more wavelengths or one or more bands of excitation wavelengths;

[0340] provide the excitation light to a detection well in the cartridge by sequentially illuminating the detection well over sub-intervals within an interrogation time interval, wherein each subinterval is correlated with a respective group of the plurality of groups such that the illumination provided to the detection well during each sub-interval comprises the one or more wavelengths or one or more bands of excitation wavelengths associated with the respective group;

[0341] generate, by a sample disposed in the detection well based on illuminating the detection well, emission light over the interrogation time interval; and

[0342] detect, by one or more photodetectors, the emission light as the plurality of fluorescent signals over the interrogation time interval.

[0343] In some aspects, the analysis cartridges are processed in an analyzer that contains software to execute one or more tasks, including the performance of the methods described herein. In some aspects, the analyzer contains software to automatically determine the next appropriate step in methods as described herein. For example, the instrument may contain software that determinesamount or presence of a target analyte of interest, such as one or more target nucleic acids. The software may display this determination, such as on a graphical user interface.

[0344] In some aspects, the analyzer stores software that instructs a processor to execute a given task. In some aspects, the software stores machine readable instructions that instruct a processor to execute a given task. The machine-readable instructions may be one or more executable programs or portion(s) of an executable program for execution by a computer. The programs may be embodied in software stored on a non-transitory computer readable storage medium such as a CD-ROM, a floppy disk, a hard drive, a DVD, a Blu-ray disk, or a memory associated with the processors. Alternatively, the entire programs and / or parts thereof could alternatively be executed by a device other than the processors and / or embodied in firmware or dedicated hardware. Additionally or alternatively, processes may be implemented by one or more hardware circuits (e.g., discrete and / or integrated analog and / or digital circuitry, an FPGA, an ASIC, a comparator, an operational-amplifier (op-amp), a logic circuit, etc.) structured to perform the corresponding operation without executing software or firmware.

[0345] The machine-readable instructions may be stored in one or more of a compressed format, an encrypted format, a fragmented format, a compiled format, an executable format, a packaged format, etc. Machine readable instructions as described herein may be stored as data (e.g., portions of instructions, code, representations of code, etc.) that may be utilized to create, manufacture, and / or produce machine executable instructions. For example, the machine-readable instructions may be fragmented and stored on one or more storage devices and / or computing devices (e.g., servers). The machine-readable instructions may require one or more of installation, modification, adaptation, updating, combining, supplementing, configuring, decryption, decompression, unpacking, distribution, reassignment, compilation, etc. in order to make them directly readable, interpretable, and / or executable by a computing device and / or other machine. For example, the machine -readable instructions may be stored in multiple parts, which are individually compressed, encrypted, and stored on separate computing devices, wherein the parts when decrypted, decompressed, and combined form a set of executable instructions that implement a program such as that described herein.

[0346] In another example, the machine-readable instructions may be stored in a state in which they may be read by a computer, but require addition of a library (e.g., a dynamic link library (DLL)), a software development kit (SDK), an application programming interface (API), etc. in order to execute the instructions on a particular computing device or other device. In another example, the machine-readable instructions may need to be configured (e.g., settings stored, data input, network addresses recorded, etc.) before the machine -readable instructions and / or thecorresponding program(s) can be executed in whole or in part. Thus, the disclosed machine-readable instructions and / or corresponding program(s) are intended to encompass such machine-readable instructions and / or program(s) regardless of the particular format or state of the machine-readable instructions and / or program(s) when stored or otherwise at rest or in transit.

[0347] The machine-readable instructions described herein can be represented by any past, present, or future instruction language, scripting language, programming language, etc. For example, the machine-readable instructions may be represented using any of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0348] The machine readable instructions may be stored on a non-transitory computer and / or machine readable medium such as a hard disk drive, a flash memory, a read-only memory, a compact disk, a digital versatile disk, a cache, a random-access memory and / or any other storage device or storage disk in which information is stored for any duration (e.g., for extended time periods, permanently, for brief instances, for temporary buffering, and / or for caching of the information). As used herein, the term non-transitory computer readable medium is expressly defined to include any type of computer readable storage device and / or storage disk and to exclude propagating signals and to exclude transmission media.

[0349] EXEMPLARY NON-LIMITING ASPECTS OF THE DISCLOSURE

[0350] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any one of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the disclosed subject matter.

[0351] 1. A system for detecting presence of a fluorescent signal in a plurality of detection wells in a cartridge, the system comprising:

[0352] a plurality of light sources operably linked to the plurality of detection wells,wherein each light source in the plurality of light sources is configured for emitting an excitation light comprising specified excitation wavelengths in a sequential manner to illuminate a specified detection well of the plurality of detection wells;

[0353] one or more photodetectors;

[0354] a conduit for transmitting an emission light comprising emission wavelengths emitted from the plurality of detection wells to one of the one or more photodetectors; and

[0355] a processor for correlating the emission light detected by the photodetector to the detection well emitting the emission light.

[0356] 2. The system according to aspect 1, wherein the plurality of light sources are arranged on a single vertical plane and the plurality of detection wells are arranged on a single horizontal plane or vice versa; wherein the plurality of light sources are arranged on a single vertical plane and the plurality of detection wells are arranged on a single vertical plane and wherein the two single vertical planes are the same vertical planes or are separate vertical plains parallel to each other; or wherein the plurality of light sources are arranged on a single horizontal plane and the plurality of detection wells are arranged on a single horizontal plane and wherein the two single horizontal planes are the same horizontal planes or are separate horizontal plains parallel to each other.

[0357] 3. The system according to aspect 1, wherein the conduit comprises a plurality of light conduits each configured to transmit light from one of the plurality of detection wells.

[0358] 4. The system according to aspect 3, wherein the light conduits are light pipes, optionally, wherein each of the light pipes comprises a length extending between an entrance point at which light enters the light pipe and an exit point at which the light exits the light pipe, at least one linear segment and at least one curved segment along the length, a tapered shape wherein a diameter of the light pipe changes, wherein the at least one linear segment comprises the tapered shape and / or wherein the at least one curved region comprises the tapered shape.

[0359] 5. The system according to aspect 1 or 2, wherein the light conduits comprise a plurality of optical fibers.

[0360] 6. The system according to any one of aspects 1-5, wherein the light sources of the plurality of light sources are arranged in a linear row.7. The system according to any one of aspects 1-6, wherein the plurality of light sources comprise “m” number of light sources that are configured to illuminate “n” number of detection wells, wherein m:n is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:2, 3:1, 3:4, 3:5, 4:1, 4:3, 4:5, 5:1, 5:2, 5:3, or 5:4, optionally such that one light source can illuminate 1 detection well, two light sources can illuminate 1 detection well, three light sources can illuminate 1 detection well, or four light sources can illuminate 1 detection well.

[0361] 8. The system according to any one of aspects 1 -7, wherein there is a 1 to 1 co-relation between the number of light sources and the number of detection wells and the system comprises at least 4 light sources and at least 4 detection wells, e.g., at least 8 light sources that are configured to illuminate at least 8 detection wells; at least 12 light sources that are configured to illuminate at least 12 detection wells; or at least 16 light sources that are configured to illuminate at least 16 detection wells.

[0362] 9. The system according to any one of aspects 1-8, wherein each light source is capable of emitting excitation light of at least two different excitation wavelengths.

[0363] 10. The system according to any one of aspects 1-7, wherein the plurality of light sources comprises: at least 8 light sources that are configured to illuminate at least 4 detection wells; at least 12 light sources that are configured to illuminate at least 6 detection wells; at least 16 light sources that are configured to illuminate at least 8 detection wells, wherein there is a 2 to 1 co-relation.

[0364] 11. The system according to any one of aspects 1-7, wherein the plurality of light sources comprises at least 32 light sources configured to illuminate at least 16 detection wells, wherein there is a 2 to 1 co-relation.

[0365] 12. The system according to aspects 8-11, wherein the light sources are arranged in a linear array, where a mthlight source illuminates the nth detection well, wherein m:n is 1:1, 1:2, 1:3, 1:4, 1:5. 2:1, 2:3, 2:5, 3:2, 3:1, 3:4, 3:5, 4:1, 4:3, 4:5. 5:1, 5:2, 5:3, or 5:4.

[0366] 13. The system according to aspects 8-12, wherein the light sources are arranged in pairs in a linear array, where a first light source and a second light source in each pair illuminates the same detection well and wherein the first light source is capable of emitting excitation light comprising excitation wavelengths different from excitation light emitted by the second light source or wherein the light sources are arranged in groups in a linear array, where a first lightsource, a second light source, and a third light source in each group illuminates the same detection well with a first excitation light, a second excitation light, and a third excitation light, respectively.

[0367] 14. The system according to any one of aspects 1-13, wherein the photodetector is a single photodetector.

[0368] 15. The system according to any one of aspects 1-14, wherein the light sources are light emitting diodes (LEDs).

[0369] 16. A device for detecting presence of a fluorescent signal in a plurality of detection wells in a cartridge, the device comprising:

[0370] a cartridge staging region for positioning the cartridge relative to the light source; and the system of any one of aspects 1-15.

[0371] 17. The device according to aspect 16, further comprising a cartridge positioned in the cartridge staging area.

[0372] 18. The device according to aspect 17, further comprising a sample present in at least one of the plurality of detection wells.

[0373] 19. The device according to aspect 18, wherein the sample comprises a positive control comprising a first fluorescent component and, if present, a second fluorescent component indicative of presence of an analyte in the sample, wherein the first and second fluorescent components have distinct fluorescence profiles.

[0374] 20. A semi-automated or automated method for detecting fluorescence in a plurality of detection wells in a cartridge in the device of any one of aspects 16-19, comprising steps of:

[0375] (a) generating an excitation light, at a specified excitation wavelength, from a mlhlight source of the plurality of light sources; illuminating a nth detection well of the plurality of detection wells; and ceasing generating the excitation light:

[0376] (b) transmitting the emission wavelength emitted from the nth detection well to the photodetector;

[0377] (c) detecting the emission wavelength on the photodetector;(d) correlating the emission wavelength detected by photodetector to the nth detection well emitting the emission wavelength, and

[0378] (e) repeating steps (a) to (d) a plurality of times to detect fluorescence in the plurality of detection wells, such that m+1 light source illuminates n+1 detection well and the photodetector correlates the detected emission wavelength to the n+1 detection well; m+2 light source illuminates n+2 detection well and the photodetector correlates the detected emission wavelength to the n+2 detection well; and so on.

[0379] 21. The method of aspect 20, wherein the plurality of light sources comprises “m” light sources, where each light source is capable of illuminating “n” well of the plurality of detection wells, wherein light source emits an excitation light at different wavelengths and the method comprises generating an excitation light from a mthlight source and illuminating the nth detection well of the plurality of detection wells and ceasing generating the excitation light and conducting steps (b) to (d) and generating an excitation light from (m+1)thlight source and illuminating the (n+1) detection well of the plurality of detection wells and ceasing generating the excitation light and conducting steps (b) to (d); or

[0380] wherein the plurality of light sources comprises a “m” and a “m+1” light source capable of illuminating “n” well of the plurality of detection wells, wherein light source emits an excitation light at different wavelengths and the method comprises generating an excitation light from a mlhand (m+1)’11light source and illuminating the nth detection well of the plurality of detection wells and ceasing generating the excitation light and conducting steps (b) to (d).

[0381] 22. The method of aspect 21, wherein the correlating comprises information regarding the light source used for generating the excitation light.

[0382] 23. The system according to aspects 21 or 22, wherein m:n correlation between each light source and each detection well is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:2, 3:1, 3:4, 3:5, 4:1, 4:3, 4:5, 5:1, 5:2, 5:3, or 5:4.

[0383] 24. The method of any one of aspects 21-23, wherein the conduit comprises a plurality of light conduits, wherein there is a 1: 1 correlation between each light conduit and each detection well and the correlating comprises information regarding the light conduit transmitting the emission wavelength to the photodetector.25. A system for detecting presence of a fluorescent signal in a plurality of detection wells in a cartridge, the system comprising:

[0384] a plurality of light sources arranged on a single plane;

[0385] an excitation filter positioned adjacent to the plurality of light sources;

[0386] a dichroic beam splitter arranged at an angle ranging from 30-60 degrees with reference to the single plane;

[0387] a conduit for interrogating one of a plurality of predetermined detection wells;

[0388] one or more photodetectors; and

[0389] a processor for correlating light detected by one of the photodetectors to one of the plurality of detection wells;

[0390] wherein:

[0391] each light source in the plurality of light sources is configured for emitting an excitation light at a specified excitation wavelength in a sequential manner to illuminate a specified detection well of the plurality of detection wells such that one detection well is illuminated at a time; the excitation filter is configured for transmitting the specified excitation wavelength towards the dichroic beam splitter;

[0392] the dichroic beam splitter is configured for reflecting the specified excitation wavelength to the conduit and for transmitting an emission wavelength received from the conduit to the one of the photodetectors;

[0393] the photodetector is configured for detecting the emission wavelength received from the detection well; and

[0394] the processor correlates the emission wavelength detected by photodetector to the detection well emitting the emission wavelength.

[0395] 26. The system according to aspect 25, wherein the conduit comprises a plurality of light conduits each configured to transmit light to one of the plurality of detection wells.27. The system according to aspect 25, wherein the conduit comprises a dynamic steerable mirror configured to sequentially reflect light to one of the plurality of detection wells.

[0396] 28. The system according to any one of aspects 25-27, wherein the plurality of light sources comprises: at least 8 light sources that are configured to illuminate at least 8 detection wells; at least 12 light sources that are configured to illuminate at least 12 detection wells; at least 16 light sources that are configured to illuminate at least 16 detection wells, wherein there is a 1 to 1 co-relation.

[0397] 29. The system according to aspect 28, wherein each light source is capable of emitting an excitation light at least two different excitation wavelengths.

[0398] 30. The system according to any one of aspects 25-29, wherein the plurality of light sources comprises “m” sources that are configured to illuminate “n” detection wells, wherein m:n is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:2, 3:1, 3:4, 3:5, 4:1, 4:3, 4:5, 5:1, 5:2, 5:3, or 5:4.

[0399] 31. The system according to any one of aspects 25-30, wherein the plurality of light sources comprises: at least 8 light sources that are configured to illuminate at least 4 detection wells; at least 12 light sources that are configured to illuminate at least 6 detection wells; at least 16 light sources that are configured to illuminate at least 8 detection wells, wherein there is a 2 to 1 co-relation.

[0400] 32. The system according to any one of aspects 25-30, wherein the plurality of light sources comprises at least 32 light sources configured to illuminate at least 16 detection wells, wherein there is a 2 to 1 co-relation.

[0401] 33. The system according to aspects 25-30, wherein the light sources are arranged in a linear array, where a m111light source illuminates the nth detection well, wherein m:n is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:2, 3:1, 3:4, 3:5, 4:1, 4:3, 4:5, 5:1, 5:2, 5:3, or 5:4.

[0402] 34. The system according to aspects 25-30, the light sources are arranged in pairs in a linear array, where a first light source and a second light source in each pair illuminates the same detection well and wherein the first light source is capable of emitting an excitation light at an excitation wavelength different from excitation light emitted by the second light source.

[0403] 35. The system according to aspect 34, wherein the photodetector is a single photodetector.36. The system according to any one of aspects 25-35, wherein the light sources are light emitting diodes (LEDs).

[0404] 37. The system according to any one of aspects 25-36, wherein the plurality of light sources are arranged in a circular configuration.

[0405] 38. The system according to any one of aspects 25-37, wherein the plurality of light sources are arranged in a concentric configuration.

[0406] 39. The system according to aspect 38, wherein an outer circle of the concentric configuration comprises at least 12 light sources and the inner circle of the concentric configuration comprises at least 4 light sources.

[0407] 40. The system according to any one of aspects 25-39, wherein the photodetector is on a plane substantially perpendicular to the single plane.

[0408] 41. The system according to any one of aspects 25-39, wherein the photodetector is on the same single plane as the plurality of light sources.

[0409] 42. The system according to aspect 41, further comprising a catadioptric system. 43. The system according to aspect 42, wherein the catadioptric system comprises of an emission focus lens and a parabolic focus mirror.

[0410] 44. The system according to any one of aspects 23-43, wherein the conduit comprises light conduits or steerable mirror.

[0411] 45. The system according to aspect 44, wherein the light conduits comprise of a plurality of light pipes, optionally, wherein each light pipe comprises a length between an entrance point at which light enters the light pipe and an exit point at which the light exits the light pipe, at least one linear segment and at least one curved segment along the length, a tapered shape wherein a diameter of the light pipe changes, wherein the at least one linear segment comprises the tapered shape and / or wherein the at least one curved region comprises the tapered shape.

[0412] 46. The system according to aspect 44, wherein the light conduits comprise of a plurality of optical fibers.

[0413] 47. A device for detecting presence of a fluorescent signal in a plurality of detection wells in a cartridge, the device comprising:a cartridge staging region for positioning the cartridge: and the system of any one of aspects 26-46.

[0414] 48. The device according to aspect 47, further comprising a cartridge positioned in the cartridge staging area.

[0415] 49. The device according to aspect 48, further comprising a sample present in at least some of the plurality of detection wells.

[0416] 50. The device according to aspect 49, wherein the sample comprises a positive control comprising a first fluorescent component and, if present, a second fluorescent component indicative of presence of an analyte in the sample, wherein the first and second fluorescent components have distinct fluorescence profiles.

[0417] 51. A semi-automated or automated method of detecting fluorescence in a plurality of detection wells in a cartridge in the device of any one of aspects 47-50, comprising steps of: generating an excitation light from a plurality of light sources at specified excitation wavelengths in a sequential manner such that one of a plurality of detection wells is illuminated at a time;

[0418] directing the specified excitation wavelengths to a dichroic beam splitter such that it is reflected to a conduit / system to interrogate one of the plurality of detection wells;

[0419] illuminating a sample in one of the plurality of the detection wells with the specified excitation wavelengths to generate emission wavelengths;

[0420] directing the emission wavelengths received from the detection well to the dichroic beam splitter such that it is transmitted to a one or more photodetectors;

[0421] detecting the emission wavelengths in one of the photodetectors;

[0422] corelating the emission wavelengths detected by the photodetector to the detection well emitting the emission wavelengths.

[0423] 52. A system for detecting presence of a fluorescent signal in a plurality of detection wells in a cartridge, the system comprising;

[0424] a plurality of light sources operably linked to the plurality of detection wells,wherein a first light source in the plurality of light sources is configured for emitting an excitation light comprising specified excitation wavelengths to a first detection well and a second light source in the plurality of light sources is configured for emitting the excitation light comprising specified excitation wavelengths to a second detection well, wherein the light sources emit the excitation light in a sequential manner;

[0425] a normalization photodetector configured for detecting the excitation light from the first and the second detection wells;

[0426] a processor configured for comparing the intensity of the excitation light from the first and the second detection wells;

[0427] a first conduit for transmitting an emission light comprising emission wavelengths emitted from the first detection well to a fluorescence photodetector for detecting the emission light and a second conduit for transmitting an emission light comprising emission wavelengths emitted from the second detection well to a fluorescence photodetector, wherein the fluorescence photodetector is a single photodetector or separate photodetectors,

[0428] wherein when the intensities of the excitation light from the first and the second detection wells measured by the normalization photodetector are different, the processor adjusts the intensity of one or both of the emission lights measured at the fluorescence photodetector to compensate for the difference and / or the processor adjusts the intensity of one or both light sources such that both light sources emit excitation light of the same intensity.

[0429] 53. The system of aspect 52, wherein the system comprises a single fluorescence photodetector for sequentially measuring the emission light from the plurality of wells.

[0430] 54. The system of aspect 52 or 53, wherein the first light source comprises at least two or at least four separate light sources all configured for emitting different excitation lights and associated with the first detection well and configured such that the two or four light sources sequentially illuminate the first detection well and wherein the second light source comprises at least two or at least four separate light sources all configured for emitting different excitation lights and associated with the second detection well and configured such that the two or four light sources sequentially illuminate the second detection well.

[0431] 55. The system of aspect 54, comprising a plurality of excitation light filters positioned in an optical path between each of the light sources and a corresponding detection well illuminatedby the light source, wherein the excitation light filters are configured for blocking wavelengths outside of a specified wavelength range and transmitting wavelengths within the specified wavelength range, wherein the wavelengths within the specified wavelength range are suitable for a fluorophore present or suspected to be present in the detection well.

[0432] 56. The system of aspect 55, comprising a plurality of excitation light filters positioned in an optical path between the detection wells and the one or more normalization detectors wherein the excitation light filters are configured for blocking fluorescence from the wells and for transmitting excitation light to the one or more normalization detectors.

[0433] 57. The system of aspect 56, comprising a plurality of emission light filters positioned in an optical path between the detection wells and the one or more fluorescence detectors wherein the emission light filters are configured for blocking excitation lights from the wells and for transmitting emission light to the one or more fluorescence detectors.

[0434] 58. The system of any one of aspects 52-57, wherein the detection wells are operably associated with a thermal cycler configured for maintaining temperatures in the detection wells suitable for performance of polymerase chain reaction (PCR) in the detection wells.

[0435] 59. The system of aspect 58, wherein the thermal cycler is configured for maintaining temperatures in the detection wells for denaturation of nucleic acid, annealing of primers to the nucleic acid and for extension of the primers and wherein the detection wells include a reporter that emits a fluorescence signal upon extension of the primers and wherein the thermal cycler performs a PCR program comprising multiple cycles, each cycle comprising denaturation of nucleic acid, annealing of primers to the nucleic acid and extension of the primers.

[0436] 60. The system of aspect 59, wherein the system is configured to sequentially activate the plurality of light sources to emit the excitation light during the extension of the primers.

[0437] 61. The system of aspect 60, wherein the system is configured to sequentially activate the plurality of light sources to emit the excitation light during the extension of the primers multiple times during the PCR program.

[0438] 62. An optical detection module, comprising:

[0439] a chamber;an illumination printed circuit board comprising a plurality of light sources arranged on a single plane in a circular configuration;

[0440] an illumination optical port on one side of the chamber, adjacent the illumination printed circuit board;

[0441] a dichroic beam splitter placed in the chamber at an angle ranging from 30-60 degrees with reference to the illumination printed circuit board;

[0442] a plurality of light conduits or a dynamic steerable mirror each configured to interrogate one of a plurality of detection wells; and

[0443] a light pipe optical port on another side of the chamber, perpendicular to the illumination optical port, configured for immobilizing the plurality of light conduits;

[0444] a detection optical port on another side of the chamber;

[0445] a detection printed circuit board adjacent the detection optical port and comprising one or more photodetectors, wherein the detection optical port and the a detection printed circuit board are one a plane perpendicular to the illumination printed circuit board;

[0446] wherein the plurality of light sources in the illumination printed circuit board are configured for emitting an excitation light, comprising specified excitation wavelengths, in a sequential manner such that one detection well is interrogated at a time;

[0447] the illumination optical port is configured for transmitting the excitation light;

[0448] the dichroic beam splitter is configured for reflecting the excitation light to one of the plurality of detection wells and for transmitting an emission light received from the detection well to one of the photodetectors;

[0449] the detection optical port is configured for transmitting the emission light;

[0450] the photodetector on the detection printed circuit board is configured for measuring the fluorescence from a sample based on the detected emission light.

[0451] 63. A light pipe for collimating light entering the light pipe, the light pipe comprising: a length between an entrance point at which light enters the light pipe and an exit point at which the light exits the light pipe,at least one linear segment and at least one curved segment along the length, a tapered shape wherein a diameter of the light pipe changes,

[0452] wherein the at least one linear segment comprises the tapered shape and / or wherein the at least one curved region comprises the tapered shape.

[0453] 64. The light pipe of aspect 63, wherein the light pipe comprises at least two linear regions connected by a curved region.

[0454] 65. The light pipe of aspect 63, wherein the light pipe comprises at least three linear regions and at least two curved regions, wherein the linear regions and curved regions alternate.

[0455] 66. A system of light pipes for collimating light entering the light pipes, the system comprising a plurality of light pipes, wherein each light pipe is the light pipe of any one of aspects 63-65,

[0456] wherein the system comprises a first planar support for immobilizing the entrance points of the light pipes and a second planar support for immobilizing the exit points of the light pipes.

[0457] 67. The system of aspect 66, wherein the first support is rectangular in shape and the entrance points of the light pipes are arranged in a linear row and the second support is circular in shape and the exit points of the light pipes are arranged in a concentric shape.

[0458] 68. The light pipe of any one of aspects 63-65 or the system of aspect 66 or 67, wherein the light pipe is made from a thermoplastic polymer.

[0459] 69. The light pipe or the system of aspect 68, wherein the thermoplastic polymer is polycarbonate.

[0460] EXAMPLES

[0461] As demonstrated in the above disclosure, the present disclosure has a wide variety of applications. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, volumes, etc.) but some experimental errors and deviations should be accounted for.

[0462] Example 1: Fluorescence Measurement of low concentration fluorescent dye

[0463] A system for measuring fluorescence as shown in FIG. 1 was used to measure fluorescence from 10 nm-1000 nm FAM dye. Low LED intensities (0.1 mAmp to 20 mAmp) were used to excite the FAM dye. Integrating amp with 10pF capacitor was utilized. A high signal to noise ratio (SNR) was measured at various exposures and low LED intensities using a single photodiode detector. See FIG. 7.

[0464] Example 2: Design of Collimating Light Pipe

[0465] To make a practical, low-cost fluorimeter with multiple optical paths, several light path elements were developed.

[0466] Shown in FIG. 8A is a light pipe array that transmits light from a linear set of locations to a circular set of locations. This design is particularly useful as the light beams can be subsequently processed with radially symmetric optical elements such as focusing lenses or mirrors. By focusing these discrete ray bundles, a common photodetector can be used to measure the combined or individual optical powers onto a common photodetector for low-cost.

[0467] FIG.8B shows a light pipe design with contours to provide specific function. This device receives and transmits light along its length using total internal reflection to contain the light rays within its boundaries using Snell’ s Law. The device is placed in an air environment with an optical index of 1.0. The pipe is made from a low-cost injectable material with good optical properties (e.g., polycarbonate) which has a higher index (polycarbonate has n=1.59). If optical waves arrive at a material interface where the second medium has a higher wave speed (i.e., lower refractive index) than the first and the angle of incidence is sufficiently oblique, the wave is completely reflected back into the first medium. The angles below where this oblique angle reflects these waves is defined as the critical angle and where the condition is satisfied, total internal reflection results. The critical angle is defined as Φc = arcsin(n2 / n1). For polycarbonate, the critical angle is 39 degrees.

[0468] A straight light pipe can be designed to shape a collimated beam output by tapering the design where a larger output diameter produces a lower angular extent as the light rays exit the pipe. This can be optimized by setting up a merit function where the sin of the rays along theoptical axis are maximized at each segment. By iterating on many shapes, the design can be optimized for collimating performance. An optimized collimating straight light pipe is shown with ray tracing in FIG. 8B.

[0469] To develop an array of light pipes with collimating exit features where translation in the pattern (i.e., from linear to circular), a combination of tapering and bending is needed. Care was taken to maintain the light rays to less than the critical angle so light is contained in the light pipe for efficient power transfer. As shown in FIG. 8C, a curved and tapered light pipe is shown with good collimation using the same iterative design approach where the length of the pipe is segmented and each segment is iteratively solved to maximize the merit functions of the sine function along the optical axis and maximizing overall optical power throughput. Generally, this occurs when minimal tapering is performed in lengths where the radius of curvature are lowest.

[0470] While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the discloses subject matter. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. CLAIMSWhat is claimed is:

1. A method for detecting presence of a plurality of fluorescent signals in a detection well, the method comprising:generating, by one or more light sources, excitation light comprising one or more wavelengths or one or more bands of excitation wavelengths, wherein the one or more or one or more bands of excitation wavelengths are generated in a plurality of groups such that each group comprises or one or more wavelengths or one or more bands of excitation wavelengths;providing the excitation light to the detection well by sequentially illuminating the detection well with the plurality of groups of one or more excitation wavelengths or one or more bands of excitation wavelengths over sub-intervals within an interrogation time interval, wherein an excitation light comprising one group of one or more wavelengths or one or more bands of wavelengths is provided to the detection well over each sub-interval such that the illumination provided to the detection well during each sub-interval comprises an excitation light comprising one group of excitation wavelengths or bands of excitation wavelengths;generating, by a sample disposed in the detection well based on illuminating the detection well, emission light over the interrogation time interval, wherein the emission light comprises a plurality of groups of emission wavelengths, wherein the plurality of groups of emission wavelengths correspond to the plurality of groups of excitation wavelengths and the emission of each group of excitation wavelengths is separated by the sub-interval over the interrogation time interval; anddetecting, by one or more photodetectors, the emission light as the plurality of fluorescent signals over the interrogation time interval.

2. The method of claim 1, wherein the emission light comprises wavelengths or bands of emission wavelengths respectively correlated with the one or more wavelengths of one or more bands of excitation wavelengths provided to the detection well, andwherein the emission light is generated by the sample over the interrogation time interval, the generation further comprises sequentially emitting, during each sub-interval, the wavelengths or bands of emission wavelengths correlated with the respective wavelengths or bands of excitation wavelengths and associated with the respective group.

3. The method of claim 1 or 2, wherein detecting the emission light further comprises:separating the emission wavelengths generated by the sample by selectively filtering the emission light within each group of the plurality of groups; anddirecting the wavelengths or bands of emission wavelengths of the emission light associated with each group to a separate photodetector of the one or more photodetectors for detection, wherein each wavelength or band of emission wavelengths is detected as a corresponding fluorescent signal of the plurality of fluorescent signals.

4. The method of any one of claims 1 to 3, further comprising:amplifying, by one or more amplifiers, the plurality of fluorescent signals detected by the one or more photodetectors:multiplexing, by a multiplexer, the plurality of amplified fluorescent signals; and converting, by an analog-to-digital converter, the multiplexed plurality of amplified fluorescent signals into a digital stream.

5. The method of any of claims 1 to 4, further comprising:normalizing or adjusting, by a normalization photodetector, an intensity of the excitation light generated by the one or more light sources based on sampling at least a portion of the excitation light.

6. The method of any of claims 1 to 5, wherein at least one group of the plurality of groups comprises a plurality of wavelengths or a plurality of bands of excitation wavelengths.

7. The method of claim 6, wherein at least two of the plurality wavelengths or the plurality of bands of excitation wavelengths associated with the at least one group are simultaneously provided to the detection well during the sub-interval associated with the at least one group.

8. The method of any one of claims 1 to 7, wherein the one or more light sources comprise “n” light sources configured to generate “n” bands of excitation wavelengths.

9. The method of claim 8, wherein the “n” light sources are at least partially integrated into one or more light source components.

10. The method of any one of claims 8 or 9, wherein the plurality of groups comprises “g” groups so that the “n” bands of excitation wavelengths are distributed into the “g” groups.

11. The method of claim 10, wherein generating the excitation light further comprises sequentially activating the “g” groups of the “n” light sources over “g” sub-intervals within the interrogation time interval.

12. The method of claim 10, wherein a g:n ratio between the number of groups and the number of light sources is 1:1, 1:2, 1:3, 1:4, 2:3, 2:5, 2:7, 3:4, 3:5, 3:7, or 3:8.

13. The method of any one of claims 1 to 12, wherein generating the excitation light further comprises, during a sub-interval within the interrogation time interval, simultaneously activating each light source associated with a group corresponding to the sub-interval.

14. The method of any one of claims 1 to 13, wherein each band of excitation wavelengths comprises one or more excitation wavelengths, and wherein each band of emission wavelengths comprises one or more emission wavelengths.

15. The method of any one of claims 1 to 14, wherein the sub-intervals within the interrogation time interval are non-overlapping in time.

16. The method of any one of claims 1 to 15, wherein the distribution of the bands of excitation wavelengths into the plurality of groups is based on one or more of: (a) increasing a spectral separation of the wavelengths or bands of excitation wavelengths; (b) increasing a spectral separation of the wavelengths or bands of emission wavelengths; or (c) increasing a signal -to-noise ratio of detection.

17. The method of any one of claims 1 to 16, wherein eight wavelengths or eight bands of excitation wavelengths are distributed into three groups.

18. The method of claim 17, wherein a first group of the three groups comprises a first, a fourth, and a sixth wavelength or band of excitation wavelengths: wherein a second group of the three groups comprises a second, a fifth, and an eighth wavelength or band of excitationwavelengths; and wherein a third group of the three groups comprises a third and a seventh wavelength or band of excitation wavelengths.

19. The method of claim 18, wherein the bands of excitation wavelengths are numbered based on arranging the eight wavelengths or bands of excitation wavelengths in order of increasing wavelength.

20. The method of any one of claims 1 to 19, wherein one or more steps of the method associated with detecting the presence of a plurality of fluorescence signals in a detection well are applied to a plurality of detection wells.

21. A system for detecting presence of a plurality of fluorescent signals in a detection well, the system comprising:one or more light sources configured to generate an excitation light comprising one or more wavelengths or one or more bands of excitation wavelengths, wherein the one or more wavelengths or one or more bands of excitation wavelengths are distributed into a plurality of groups such that each group comprises one or more wavelengths or one or more bands of excitation wavelengths;a first conduit for transmitting the excitation light to the detection well, wherein the one or more light sources are configured to sequentially illuminate the detection well with the plurality of groups of one or more wavelengths or one or more bands of excitation wavelengths over subintervals within an interrogation time interval, wherein an excitation light comprising one group of excitation wavelengths or bands of excitation wavelengths is provided to the detection well over each sub-interval such that the illumination transmitted to the detection well during each subinterval comprises excitation light comprising one group of excitation wavelengths or one or more bands of excitation wavelengths;one or more photodetectors; anda second conduit for transmitting, to the one or more photodetectors, emission light generated by a sample disposed in the detection well over the interrogation time interval.

22. The system of claim 21, wherein the emission light comprises one or more wavelengths or one or more bands of emission wavelengths respectively correlated with the one or more wavelengths or one or more bands of excitation wavelengths provided to the detection well, andwherein the emission light is generated by the sample over the interrogation time interval, the generation further comprises sequentially emitting, during each sub-interval, the wavelengths or bands of emission wavelengths correlated with the respective wavelengths or bands of excitation wavelengths and associated with the respective group.

23. The system of claim 21 or 22, wherein a waveguide is configured as the first conduit to transmit the excitation light to the detection well.

24. The system of claim 23, wherein the waveguide is further configured as the second conduit to transmit the emission light away from the detection well.

25. The system of any one of claims 21 to 24, further comprising one or more filters configured to separate the bands of emission wavelengths generated by the sample by selectively filtering the emission light within each group of the plurality of groups.

26. The system of any one of claims 21 to 25, further comprising a plurality of photodetectors,wherein the system is configured to direct the one or more emission wavelengths produced from one or more excitation wavelengths within each group to a photodetector of the one or more photodetectors for detection, andwherein each wavelength or band of emission wavelengths is detected as a corresponding fluorescent signal of the plurality of fluorescent signals.

27. The system of any one of claims 21 to 26, further comprising one or more amplifiers configured to amplify the plurality of fluorescent signals detected by the one or more photodetectors.

28. The system of claim 27, further comprising a multiplexer configured to multiplex the plurality of amplified fluorescent signals.

29. The system of claim 28, further comprising an analog-to-digital converter configured to convert the multiplexed plurality of amplified fluorescent signals into a digital stream.

30. The system of any one of claims 21 to 29, further comprising a normalization photodetector configured to normalize or adjust an intensity of the excitation light generated by the one or more light sources based on sampling at least a portion of the excitation light.

31. The system of any one of claims 21 to 30, wherein at least one group of the plurality of groups comprises a plurality of wavelengths or a plurality of bands of excitation wavelengths.

32. The system of claim 31, wherein at least two of the plurality of wavelengths or the plurality of bands of excitation wavelengths associated with the at least one group are simultaneously provided to the detection well during the sub-interval associated with the at least one group.

33. The system of any one of claims 21 to 32, wherein the one or more light sources comprise “n” light sources configured to generate “n” wavelengths or “n” bands of excitation wavelengths.

34. The system of claim 33, wherein the “n” light sources are at least partially integrated into one or more light source components.

35. The system of any of claims 33 or 32, wherein the plurality of groups comprises “g” groups so that the “n” bands of excitation wavelengths are distributed into the “g” groups.

36. The system of claim 35, wherein generating the excitation light further comprises sequentially activating the “g” groups of the “n” light sources over “g” sub-intervals within the interrogation time interval.

37. The system of claim 35, wherein a g:n correlation between the number of groups and the number of light sources is 1:1, 1:2, 1:3, 1:4, 2:3, 2:5, 2:7, 3:4, 3:5, 3:7, or 3:8.

38. The system of any one of claims 21 to 37, wherein generating the excitation light further comprises, during a sub-interval within the interrogation time interval, simultaneously activating each light source associated with a group corresponding to the sub-interval.

39. The system of any one of claims 21 to 38, wherein each group of excitation wavelengths comprises one or more excitation wavelengths, and wherein each group of emission wavelengths comprises one or more emission wavelengths.

40. The system of any one of claims 21 to 39, wherein the sub-intervals within the interrogation time interval are non-overlapping in time.

41. The system of any one of claims 21 to 40, wherein the distribution of the wavelengths or bands of excitation wavelengths into the plurality of groups is based on one or more of: (a) increasing a spectral separation of the wavelengths or bands of excitation wavelengths; (b) increasing a spectral separation of the wavelengths or bands of emission wavelengths; or (c) increasing a signal-to-noise ratio of detection.

42. The system of any one of claims 21 to 41, comprising eight frequencies of excitation wavelengths distributed into three groups.

43. The system of claim 42, wherein a first group of the three groups comprises a first, a fourth, and a sixth wavelength or band of excitation wavelengths; wherein a second group of the three groups comprises a second, a fifth, and an eighth wavelength or band of excitation wavelengths; and wherein a third group of the three groups comprises a third and a seventh wavelength or band of excitation wavelengths.

44. The system of claim 43, wherein the wavelengths or bands of excitation wavelengths are numbered based on arranging the eight wavelengths or bands of excitation wavelengths in order of increasing wavelength.

45. The method of any one of claims 1 to 16 and 21-41, wherein the one or more light sources comprises five light sources.

46. The method of any one of claims 1 to 16, 20 and 45, wherein the emission light comprises six wavelengths or bands of emission light.

47. The method of any one of claims 21 to 41, and 46, wherein the emission light comprises six wavelengths or bands of emission light.

48. A method for detecting presence of a plurality of fluorescent signals in a detection well, the method comprising:generating, by five light sources, excitation light comprising one or more wavelengths or one or more bands of excitation wavelengths, wherein the one or more wavelengths or one or more bands of excitation wavelengths are distributed into a plurality of groups such that each group comprises one or more wavelengths or one or more bands of excitation wavelengths; providing the excitation light to the detection well by sequentially illuminating the detection well with the plurality of groups of one or more wavelengths or one or more bands of excitation wavelengths over sub-intervals within an interrogation time interval, wherein an excitation light comprising one group of one or more wavelengths or one or more bands of excitation wavelengths is provided to the detection well over each sub-interval such that the illumination provided to the detection well during each sub-interval comprises an excitation light comprising one group of excitation wavelengths;generating, by a sample disposed in the detection well based on illuminating the detection well, six bands of emission light over the interrogation time interval, wherein the emission light comprises a plurality of groups of emission wavelengths, wherein the plurality of groups of emission wavelength correspond to the plurality of groups of excitation wavelengths and the emission of each group of excitation wavelengths is separated by the sub-interval over the interrogation time interval; anddetecting, by one or more photodetectors, the six bands of emission light as the plurality of fluorescent signals over the interrogation time interval.

49. The method of claim 48, wherein the bands of emission light comprise bands of emission wavelengths respectively correlated with the bands of excitation wavelengths provided to the detection well, andwherein the emission light is generated by the sample over the interrogation time interval, the generation further comprises sequentially emitting, during each sub-interval, the bands of emission wavelengths correlated with the respective bands of excitation wavelengths and associated with the respective group.

50. The method of claim 48 or 49, wherein detecting the emission light further comprises:separating the emission wavelengths generated by the sample by selectively filtering the emission light within each group of the plurality of groups; anddirecting the bands of emission wavelengths of the emission light associated with each group to a separate photodetector of the one or more photodetectors for detection, wherein each band of emission wavelengths is detected as a corresponding fluorescent signal of the plurality of fluorescent signals.

51. The method of any one of claims 48-50, further comprising:amplifying, by one or more amplifiers, the plurality of fluorescent signals detected by the one or more photodetectors:multiplexing, by a multiplexer, the plurality of amplified fluorescent signals; and converting, by an analog-to-digital converter, the multiplexed plurality of amplified fluorescent signals into a digital stream.

52. The method of any one of claims 48-51, further comprising:normalizing or adjusting, by a normalization photodetector, an intensity of the excitation light generated by the one or more light sources based on sampling at least a portion of the excitation light.

53. The method of any one of claims 48-52, wherein at least one group of the plurality of groups comprises a plurality of wavelengths or a plurality of bands of excitation wavelengths.

54. The method of claim 53, wherein at least two of the plurality wavelengths or the plurality of bands of excitation wavelengths within the at least one group are simultaneously provided to the detection well during the sub-interval associated with the at least one group.

55. The method of any one of claims 48-54, wherein the five light sources are at least partially integrated into one or more light source components.

56. The method of any one of claims 48-55, wherein generating the excitation light further comprises sequentially activating the three groups of the five light sources over three subintervals within the interrogation time interval.

57. The method of any one of claims 48-56, wherein generating the excitation light further comprises, during a sub-interval within the interrogation time interval, simultaneously activating each light source associated with a group corresponding to the sub-interval.

58. The method of any one of claims 48-57, wherein each group of excitation wavelengths comprises one or more excitation wavelengths, and wherein each group of emission wavelengths comprises one or more emission wavelengths.

59. The method of any of claims 48-58, wherein the sub-intervals within the interrogation time interval are non-overlapping in time.

60. The method of any one of claims 48-59, wherein the distribution of the excitation wavelengths into the plurality of groups is based on one or more of: (a) increasing a spectral separation of the bands of excitation wavelengths: (b) increasing a spectral separation of the bands of emission wavelengths; or (c) increasing a signal-to-noise ratio of detection.

61. The method of any one of claims 48-60, wherein the plurality of groups comprises three groups so that the five bands of excitation wavelengths are distributed into the three groups.

62. The method of claim 61, wherein a first group of the three groups comprises a first wavelength or a first band of excitation wavelength; wherein a second group of the three groups comprises a third and fourth wavelength or a fourth excitation wavelengths; and wherein a third group of the three groups comprises a second and a fifth wavelength or a fifth band of excitation wavelengths.

63. The method of any one of claim 48-62, wherein the wavelengths or bands of excitation wavelengths are numbered based on arranging the five wavelengths or bands of excitation wavelengths in order of increasing wavelength.

64. The method of any one of claims 48-63, wherein one or more steps of the method associated with detecting the presence of a plurality of fluorescence signals in a detection well are applied to a plurality of detection wells.

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