# US 10,768,110 B2 · Apparatus and method for differentiating multiple fluorescence signals by excitation wavelength > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,768,110 B2 - Document ID: US10768110B2 - Status: Granted - Kind: B2 - Application number: 12/642,009 - Filed: 2009-12-18 - Published: 2010-08-26 - Granted: 2020-09-08 - Earliest claimed: 2002-05-17 - Assignees: Applied Biosystems, LLC - Applicants: Howard G. King; Steven J. Boege; Eugene F. Young; Mark F. Oldham - Inventors: Howard G. King; Steven J. Boege; Eugene F. Young; Mark F. Oldham - Web page: https://patentgrep.com/patent/US10768110B2 ## CPC - G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)} - B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples} - G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates} - B01L 2300/1805: Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks - G01N 2021/6419: {Excitation at two or more wavelengths} - G01N 2021/6421: {Measuring at two or more wavelengths} - G01N 2021/6441: {with two or more labels} - G01N 2201/062: LED's - G01N 2201/0627: Use of several LED's for spectral resolution - G01N 2201/0628: Organic LED [OLED] - Y10S 435/808: Optical sensing apparatus - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract An apparatus and method are provided for differentiating multiple detectable signals by excitation wavelength. The apparatus can include a light source that can emit respective excitation light wavelengths or wavelength ranges towards a sample in a sample retaining region, for example, in a well. The sample can contain two or more detectable markers, for example, fluorescent dyes, each of which can be capable of generating increased detectable emissions when excited in the presence of a target component. The detectable markers can have excitation wavelength ranges and/or emission wavelength ranges that overlap with the ranges of the other detectable markers. A detector can be arranged for detecting an emission wavelength or wavelength range emitted from a first marker within the overlapping wavelength range of at least one of the other markers. ## Claims ### Claim 1 An apparatus comprising: a sample well holding assembly configured to hold an array of sample wells, each sample well configured to receive a sample containing a nucleic acid sequence and a plurality of different detectable markers; a plurality of excitation light sources, each excitation light source capable of emitting a plurality of wavelength ranges, each of the plurality of excitation wavelength ranges corresponding to a respective excitation wavelength range of a respective one of the plurality of different detectable markers; a plurality of detectors, each of the detectors capable of detecting an emission wavelength range emitted from each of the plurality of detectable markers; a plurality of lenses, each of the lenses configured to direct excitation light from one of the excitation light sources into a respective one of the sample wells and to receive emission light from the respective one of the sample wells; a plurality of lightpipes, each lightpipe having one of the lenses at a first end of the lightpipe, and a second end proximal a respective one of the plurality of excitation light sources and a respective one of the detectors, wherein light from the respective light source and emission light from the respective sample well pass through a common cross-section of the second end. ### Claim 2 (depends on 1) The apparatus of claim 1, further comprising at least two lenses configured to direct the emission beams from the array of sample wells to the detector. ### Claim 3 (depends on 1) The apparatus of claim 1, further comprising an excitation wavelength-excluding device disposed along an emission beam path between at least one of the sample wells and the detector. ### Claim 4 (depends on 3) The apparatus of claim 3, wherein the excitation wavelength-excluding device comprises a filter located along the emission beam path between at least one of the sample wells and a lens configured to direct emission beams to the detector. ### Claim 5 (depends on 1) The apparatus of claim 1, wherein the detector is capable of comparing an emission beam corresponding to each of the excitation light sources to determine the presence or absence of a component. ### Claim 6 (depends on 1) The apparatus of claim 1, wherein the plurality of excitation light sources comprises an array of light emitting lasers. ### Claim 7 (depends on 1) The apparatus of claim 1, wherein the detector comprises a first photodiode detector configured to receive emission light produced by a first excitation beam of the plurality of excitation light sources and a second photodiode detector configured to receive emission light produced by a second excitation beam of the plurality of excitation light sources. ### Claim 8 (depends on 1) The apparatus of claim 1, wherein the detector comprises at least one of a charge coupled device. ### Claim 9 (depends on 1) The apparatus of claim 1, wherein the detector comprises at least one of a CMOS detector. ### Claim 10 (depends on 1) The apparatus of claim 1, wherein the plurality of excitation light sources comprises a plurality of light emitting diodes. ### Claim 11 (depends on 1) The apparatus of claim 1, further comprising a second plurality of lenses, each lens of the second plurality of lenses disposed between a respective one of the detectors and a respective end of one of the lightpipes. ### Claim 12 (depends on 1) The apparatus of claim 1, wherein each lens of the plurality of lenses is disposed proximal a respective sample well of the array of sample wells. ### Claim 13 (depends on 1) The apparatus of claim 1, further comprising: one or more excitation filters configured to control one or more of the wavelength ranges of the excitation light sources; and one or more emission filters configured to control the wavelength of emissions from one or more of the sample wells. ### Claim 14 An apparatus comprising: a reaction region holding assembly configured to hold an array of reaction regions, each reaction region configured to receive a sample containing a nucleic acid sequence and a plurality of different detectable markers; a plurality of excitation light sources, each excitation light source configured to emit a plurality of wavelength ranges, each of the plurality of excitation wavelength ranges corresponding to a respective excitation wavelength range of a respective one of the plurality of different detectable markers; a plurality of detectors, each of the detectors configured to detect an emission wavelength range emitted from each of the plurality of detectable markers; a plurality of lenses, each of the lenses configured to direct excitation light from one of the excitation light sources into a respective one of the reaction regions and to direct emission light from the respective one of the reaction regions to a respective one of the detectors; a plurality of lightpipes, each lightpipe having one of the lenses at a first end of the lightpipe, and a second end proximal a respective one of the plurality of excitation light sources and a respective one of the detectors, wherein light from the respective light source and emission light from the respective reaction region pass through a common cross-section of the second end. ### Claim 15 (depends on 14) The apparatus of claim 14, wherein the plurality of detectors are capable of comparing emission wavelength ranges corresponding to each of the excitation wavelength ranges to determine the presence or absence of a component. ### Claim 16 (depends on 14) The apparatus of claim 14, wherein the plurality of excitation light sources comprises an array of light emitting diodes lasers. ### Claim 17 (depends on 14) The apparatus of claim 14, further comprising an excitation wavelength-excluding device arranged between the reaction region holding assembly and the detector. ### Claim 18 (depends on 14) The apparatus of claim 14, further comprising an excitation wavelength-excluding device arranged between the excitation light source and the reaction region holding assembly. ### Claim 19 (depends on 14) The apparatus of claim 14, wherein the detector comprise a first photodiode detector configured to receive emission light produced by a first excitation wavelength range of the plurality of excitation wavelength ranges and a second photodiode detector configured to receive emission light produced by a second excitation wavelength range of the plurality of excitation wavelength ranges. ### Claim 20 (depends on 14) The apparatus of claim 14, wherein the plurality of excitation light sources comprises a plurality of light emitting diodes. ### Claim 21 (depends on 14) The apparatus of claim 14, further comprising a second plurality of lenses, each lens of the second plurality of lenses disposed between a respective one of the detectors and a respective end of one of the lightpipes. ### Claim 22 (depends on 14) The apparatus of claim 14, wherein each lens of the plurality of lenses is disposed proximal a respective reaction region of the array of reaction regions. ### Claim 23 (depends on 14) The apparatus of claim 14, further comprising: one or more excitation filters configured to control one or more of the wavelength ranges of the excitation light sources; and one or more emission filters configured to control the wavelength of emissions from one or more of the reaction regions. ### Claim 24 An apparatus comprising: a reaction region holding assembly configured to hold a plurality of reaction regions, each reaction region configured to receive a sample containing a nucleic acid sequence and a plurality of different detectable markers; a plurality of excitation light sources, each excitation light source configured to emit a plurality of wavelength ranges; a plurality of detectors, each of the detectors configured to detect emissions from the detectable markers; a plurality of lenses, each of the lenses configured to direct excitation light from one of the excitation light sources into a respective one of the reaction regions and to direct emissions from the respective one of the reaction regions to a respective one of the detectors; a plurality of lightpipes, each lightpipe comprising: a first end proximal one of the lenses, and a second end proximal a respective one of the plurality of excitation light sources and a respective one of the detectors, wherein light from the respective light source and emissions from the respective reaction site pass through a common cross-section of the second end. ### Claim 25 (depends on 24) The apparatus of claim 24, wherein each lens of the plurality of lenses is disposed proximal a respective reaction region of the plurality of reaction regions. ### Claim 26 (depends on 24) The apparatus of claim 24, further comprising at least two lenses configured to direct the emission beams from the plurality of reaction regions to the detector. ### Claim 27 (depends on 24) The apparatus of claim 24, wherein the plurality of excitation light sources comprises a plurality of light emitting diodes. ### Claim 28 (depends on 24) The apparatus of claim 24, further comprising a second plurality of lenses, each lens of the second plurality of lenses disposed between a respective one of the detectors and a respective end of one of the lightpipes. ### Claim 29 (depends on 24) The apparatus of claim 24, further comprising: one or more excitation filters configured to control one or more of the wavelength ranges of the excitation light sources; and one or more emission filters configured to control the wavelength of emissions from one or more of the reaction regions.