# US 2019/0376894 A1 · Apparatus and methods for fluorescence imaging using radiofrequency-multiplexed excitation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0376894 A1 - Document ID: US20190376894A1 - Status: Published application - Kind: A1 - Application number: 16/528,436 - Filed: 2019-07-31 - Published: 2019-12-12 - Earliest claimed: 2013-01-09 - Assignees: The Regents of the University of California - Applicants: The Regents of the University of California - Inventors: Eric D. Diebold; Bahram Jalali; Brandon Buckley - Web page: https://patentgrep.com/patent/US20190376894A1 ## CPC - G01N 21/6408: {with measurement of decay time, time resolved fluorescence} - G01N 21/05: Flow-through cuvettes (G01N21/09 takes precedence; handling fluid samples G01N1/10) - G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)} - G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)} - G02B 21/0076: {arrangements using fluorescence or luminescence} - G02B 21/0084: {time-scale detection, e.g. strobed, ultra-fast, heterodyne detection} - G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)} - G02B 27/1006: {for splitting or combining different wavelengths (G02B27/1086, G02B27/141 take precedence)} - G02F 1/11: based on acousto-optical elements, e.g. using variable diffraction by sound or like mechanical waves (acousto-optical deflection G02F1/33) - G01N 2021/6415: {with two excitations, e.g. strong pump/probe flash} - G01N 2021/6419: {Excitation at two or more wavelengths} - G01N 2201/06113: Coherent sources; lasers - 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) - G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS - G02F: OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS ## Abstract Apparatus and methods for fluorescence imaging using radiofrequency multiplexed excitation. One apparatus splits an excitation laser beam into two arms of a Mach-Zehnder interferometer. The light in the first beam is frequency shifted by an acousto-optic deflector, which is driven by a phase-engineered radiofrequency comb designed to minimize peak-to-average power ratio. This RF comb generates multiple deflected optical beams possessing a range of output angles and frequency shifts. The second beam is shifted in frequency using an acousto-optic frequency shifter. After combining at a second beam splitter, the two beams are focused to a line on the sample using a conventional laser scanning microscope lens system. The acousto-optic deflectors frequency-encode the simultaneous excitation of an entire row of pixels, which enables detection and de-multiplexing of fluorescence images using a single photomultiplier tube and digital phase-coherent signal recovery techniques. ## Claims ### Claim at position 1 1-20. (canceled) ### Claim 21 An apparatus comprising: a light beam generator configured to generate a plurality of frequency shifted beams of light, the light beam generator comprising: a laser; an acousto-optic deflector (AOD); and an acousto-optic frequency shifter (AOFS); an optical apparatus configured to direct a first set of frequency shifted beams of light along a first optical path and to direct a second set of frequency shifted beams of light along a second optical path; and a photodetector. ### Claim 22 (depends on 21) The apparatus according to claim 21, wherein the first set of frequency shifted beams of light are generated with the acousto-optic deflector. ### Claim 23 (depends on 21) The apparatus according to claim 21, wherein the second set of frequency shifted beams of light are generated with the acousto-optic frequency shifter. ### Claim 24 (depends on 21) The apparatus according to claim 21, wherein the light beam generator further comprises a beam splitter configured to propagate a first beam of laser light to the acousto-optic deflector and a second beam of laser light to the acousto-optic frequency shifter. ### Claim 25 (depends on 21) The apparatus according to claim 21, wherein the apparatus further comprises a second optical apparatus configured to combine the first set of frequency shifted beams of light and the second set of frequency shifted beams of light. ### Claim 26 (depends on 25) The apparatus according to claim 25, wherein the second optical apparatus is configured to propagate at least one beam of light from the first set of frequency shifted beams of light along a third optical path and at least one beam of light from the second set of frequency shifted beams of light along a fourth optical path. ### Claim 27 (depends on 26) The apparatus according to claim 26, wherein the first optical apparatus comprises a non-polarizing a beam splitter. ### Claim 28 (depends on 26) The apparatus according to claim 26, wherein the second optical apparatus comprises a beam splitter. ### Claim 29 (depends on 26) The apparatus according to claim 26, further comprising a scanning mirror configured to propagate light from the second optical apparatus to a sample. ### Claim 30 (depends on 29) The apparatus according to claim 29, wherein the scanning mirror is a resonant scan galvanometer mirror. ### Claim 31 (depends on 29) The apparatus according to claim 29, wherein the photodetector is configured to detect fluorescence from the sample. ### Claim 32 (depends on 21) The apparatus according to claim 21, wherein the laser is a continuous wave laser. ### Claim 33 A method comprising: generating a plurality of frequency shifted beams of light with a light beam generator comprising: a laser; an acousto-optic deflector (AOD); and an acousto-optic frequency shifter (AOFS); propagating a first set of frequency shifted beams of light along a first optical path; propagating a second set of frequency shifted beams of light along a second optical path; combining at least one beam of light from the first set of frequency shifted beams of light and at least one beam from the second set of frequency shifted beams of light; propagating the at least one beam of light from the first set of frequency shifted beams of light and the at least one beam from the second set of frequency shifted beams of light onto a sample; and detecting one or more light signals from the sample. ### Claim 34 (depends on 33) The method according to claim 33, wherein the first set of frequency shifted beams of light are generated with the acousto-optic deflector. ### Claim 35 (depends on 33) The method according to claim 33, wherein the second set of frequency shifted beams of light are generated with the acousto-optic frequency shifter. ### Claim 36 (depends on 33) The method according to claim 33, wherein the light beam generator further comprises a beam splitter configured to propagate a first beam of laser light to the acousto-optic deflector and a second beam of laser light to the acousto-optic frequency shifter. ### Claim 37 (depends on 33) The method according to claim 33, wherein propagating light from the first set of frequency shifted beams of light and the second set of frequency shifted beams of light onto the sample comprises propagating the beams of light to the sample with a scanning mirror. ### Claim 38 (depends on 37) The method according to claim 37, wherein the scanning mirror is a resonant scan galvanometer mirror.