# US 11,307,143 B2 · Systems, apparatuses and methods for simultaneous multi-plane imaging > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,307,143 B2 - Document ID: US11307143B2 - Status: Granted - Kind: B2 - Application number: 16/900,279 - Filed: 2020-06-12 - Published: 2020-10-01 - Granted: 2022-04-19 - Earliest claimed: 2017-12-12 - Assignees: Allen Institute - Applicants: Allen Institute - Inventors: Dmitri Tsyboulski; Natalia Orlova; Jerome Anthony Lecoq; Peter Saggau - Web page: https://patentgrep.com/patent/US11307143B2 ## CPC - G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)} - G01N 21/6402: {Atomic fluorescence; Laser induced fluorescence} - G02B 21/0032: {Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers (G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06)} - G02B 21/0076: {arrangements using fluorescence or luminescence} - G02B 21/367: {providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison} - G02B 27/283: {used for beam splitting or combining} - G02B 2207/114: Two photon or multiphoton effect - 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 ## Abstract A system includes a light source to generate an optical signal having a set of pulses at a first repetition rate. The system also includes a multiplexer circuit to generate a multiplexed optical signal from the optical signal n sets of pulses at a second repetition rate, where the n sets of pulses have different polarization states and are at the first repetition rate. The system also includes a focusing unit to split the multiplexed optical signal into n excitation signals to excite a sample. The system also includes an objective to receive the n excitation signals and to illuminate the sample. The objective and the focusing unit collectively focus each excitation signal of the n excitation signals on a different focal plane of the sample to generate a response signal. The system also includes a demultiplexer circuit to generate n emission signals based on the response signal. ## Claims ### Claim 1 A system, comprising: a light source to generate an optical signal, the optical signal including a set of pulses at a first repetition rate; an optical multiplexer circuit to receive the optical signal and to generate a multiplexed optical signal including a set of pulses at a second repetition rate, wherein the second repetition rate is an n-multiple of the first repetition rate, wherein the set of pulses at the second repetition rate includes n sets of pulses, each set of pulses of the n set of pulses having a different polarization state than each other of the n set of pulses and being at the first repetition rate; a focusing unit to receive the multiplexed optical signal and to split the multiplexed optical signal into a set of n excitation signals to excite a sample, each excitation signal of the set of n excitation signals corresponding to a set of pulses of the n set of pulses; an objective to receive the set of n excitation signals and to illuminate the sample, wherein the objective and the focusing unit collectively focus each excitation signal of the set of n excitation signals on a different focal plane of the sample than each other excitation signal of the set of n excitation signals to generate a response signal in response to the set of n excitation signals; and an electronic demultiplexer circuit to demultiplex emission from the different focal planes and to generate a set of n emission signals based on the response signal, each emission signal of the set of n emission signals corresponding to an excitation signal of the set of n excitation signals. ### Claim 2 (depends on 1) The system of claim 1, wherein the objective is a first objective, the focusing unit including n sets of focusing optics, each set of focusing optics having a longitudinal axis different than the longitudinal axis of the other sets of focusing optics and setting a focus of one excitation signal in the set of n excitation signals, each set of focusing optics including: a quarter wave plate; a second objective; and a focusing mirror to independently adjust a position of the focal plane of the corresponding excitation signal. ### Claim 3 (depends on 2) The system of claim 2, wherein n=2, and wherein each set of pulses has a duty cycle of about 50%. ### Claim 4 (depends on 1) The system of claim 1, wherein n=2, the multiplexer circuit including an electro-optic modulator and a set of polarizing beam splitters to receive the optical signal and to generate the two sets of pulses as a first set of s-polarized pulses and a second set of p-polarized pulses. ### Claim 5 (depends on 1) The system of claim 1, wherein the light source is a laser source operating at a wavelength from about 700 nm to about 1000 nm, and wherein the first repetition rate is up to about 80 MHz. ### Claim 6 (depends on 1) The system of claim 1, further comprising a scanning unit in the optical path between the focusing unit and the objective, to scan each excitation signal of the set of n excitation signals in its respective focal plane in the sample. ### Claim 7 (depends on 1) The system of claim 1, further comprising a scanning unit in the optical path between the multiplexer circuit and the focusing unit, to scan each set of pulses of the n sets of pulses in its respective focal plane in the sample. ### Claim 8 (depends on 1) The system of claim 1, wherein the n sets of pulses include a first set of pulses and a second set of pulses, further comprising: a beam splitter configured to split the first set of pulses and the second set of pulses; a first scanning unit in the optical path between the beam splitter and the focusing unit, to receive the first set of pulses and to scan the first set of pulses in its respective image plane in the sample; and a second scanning unit in the optical path between the beam splitter and the focusing unit, to receive the second set of pulses and to scan the second set of pulses in its respective focal plane in the sample. ### Claim 9 (depends on 1) The system of claim 1, further comprising a detector communicably coupled to the demultiplexer circuit, to receive the response signal. ### Claim 10 (depends on 9) The system of claim 9, the multiplexer unit including a delay line to introduce a temporal separation between the n sets of pulses. ### Claim 11 (depends on 10) The system of claim 10, wherein the temporal separation is based on a) a duty cycle associated with each set of pulses of then sets of pulses, and b) a decay time associated with fluorescence emission from the sample in response to the set of n excitation signals. ### Claim 12 (depends on 9) The system of claim 9, wherein the demultiplexer circuit separates the response signal into the set of n emission signals based on a set of reference signals from the light source. ### Claim 13 (depends on 9) The system of claim 9, wherein the demultiplexer circuit includes a switch communicably coupled to the detector to generate the set of n emission signals based on a set of reference signals from the light source. ### Claim 14 A method, comprising: generating an optical signal including a set of pulses at a first repetition rate, wherein the first repetition rate is an n-multiple of a second repetition rate, wherein the set of pulses at the second repetition rate include n sets of pulses, each set of pulses of then set of pulses having a different polarization state than each other of the n set of pulses and operating at the first repetition rate; splitting the optical signal into a set of n excitation signals to excite a sample, each excitation signal of the set of n excitation signals corresponding to a set of pulses of the n set of pulses; focusing each excitation signal of the set of n excitation signals to a different focal plane of the sample than each other excitation signal of the set of n excitation signals to generate a response signal in response to the set of n excitation signals; and separating the emission from the different focal planes in the response signal into a set of n emission signals, each emission signal of the set of n emission signals corresponding to an excitation signal of the set of n excitation signals. ### Claim 15 (depends on 14) The method of claim 14, further comprising: independently adjusting a position of the focal plane of each excitation signal. ### Claim 16 (depends on 15) The method of claim 15, wherein the n sets of pulses include two sets of pulses, the generating including generating the two set of pulses as a first set of s-polarized pulses and a second set of p-polarized pulses. ### Claim 17 (depends on 14) The method of claim 14, further comprising scanning each excitation signal of the set of n excitation signals in its respective focal plane in the sample. ### Claim 18 (depends on 14) The method of claim 14, further comprising scanning each set of pulses of then sets of pulses in its respective focal plane in the sample. ### Claim 19 (depends on 14) The method of claim 14, wherein then sets of pulses include a first set of pulses and a second set of pulses, further comprising: splitting the first set of pulses and the second set of pulses; scanning the first set of pulses in its respective focal plane in the sample; and scanning the second set of pulses in its respective focal plane in the sample. ### Claim 20 (depends on 14) The method of claim 14, further comprising receiving, via a detector, the response signal. ### Claim 21 (depends on 20) The method of claim 20, the separating further comprising separating the response signal into the set of n emission signals based on a set of reference signals. ### Claim 22 (depends on 20) The method of claim 20, the separating further including generating the set of n emission signals based on a set of reference signals. ### Claim 23 A system, comprising: a laser source to generate an optical signal, the optical signal including a set of pulses at a first repetition rate; an electro-optic modulator and a polarizing beam splitter to receive the optical signal and to generate a multiplexed optical signal including a set of pulses at a second repetition rate, wherein the second repetition rate is twice the first repetition rate, wherein the set of pulses at the second repetition rate include a first set of pulses and a second set of pulses each at the first repetition rate, the first set of pulses having a different polarization state than the second set of pulses; a focusing unit to receive the multiplexed optical signal and to split the multiplexed optical signal into a first excitation signal and a second excitation signal to excite a sample, the first excitation signal corresponding to the first set of pulses, the second excitation signal corresponding to the second set of pulses, the focusing unit including a first set of focusing optics and a second set of focusing optics having a different longitudinal axis than the first set of focusing optics, each of the first set of focusing optics and the second set of focusing optics including: a quarter wave plate; a second objective; and a focusing mirror to independently adjust a position of the focal plane of its corresponding excitation signal; an objective to receive the first excitation signal and a second excitation signal and optically coupled to the sample to illuminate the sample and to generate an emission from the sample, wherein the first excitation signal is focused on a first focal plane of the sample and the second excitation signal is focused on a second focal plane of the sample that is different than the first focal plane; a detector configured to generate a response signal based on the emission from the sample; and a switch to separate the response signal into a first emission signal corresponding to the first excitation signal and a second emission signal corresponding to the second excitation signal.