# US 11,994,456 B2 · System and method to select and isolate particles and cells and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,994,456 B2 - Document ID: US11994456B2 - Status: Granted - Kind: B2 - Application number: 17/267,888 - Filed: 2019-08-16 - Published: 2021-06-10 - Granted: 2024-05-28 - Earliest claimed: 2018-08-17 - Assignees: Enrich Biosystems Inc. - Applicants: Qi Zhao; Enrich Biosystems Inc. - Inventors: Qi Zhao - Web page: https://patentgrep.com/patent/US11994456B2 ## CPC - G01N 15/1433: using image recognition - G01N 1/36: Embedding or analogous mounting of samples - G01N 15/1434: Optical arrangements - G01N 15/149: specially adapted for sorting particles, e.g. by their size or optical properties - G01N 2015/1006: {for cytology} - G01N 2015/1447: {Spatial selection} - 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) ## Abstract The present disclosure provides, among other things, methods and devices for single cells/particle selection and isolation from a sample in a light-curable biomatrix gel. The individual cells or particles are observed by microscopy and manipulated and isolated by accurately controlling the light transmission from a liquid crystal display (LCD) oriented under the sample to provide a detailed image of the sample. In some embodiments, selected cells or particles are then immobilized by curing the biomatrix gel with blue/violet light transmitted from a LED pixel array. In other embodiments, the selected cells or particles of interest are allowed to remain mobile. In either embodiment, the individual cells or particles of interest are segregated from and isolated from the remainder of the sample. ## Claims ### Claim 1 A method for segregating particles from a sample prepared in a light-curable polymeric medium, utilizing a system, comprising: a light transparent sample plate having one or more receptacles for containing the sample, one or more light sources for illuminating and/or curing the sample, a liquid crystal display (LCD) for selectively controlling the transmission of light to the sample, a sample stage for aligning the sample plate and the LCD with respect to each other, an objective for observing the sample by microscopy, a camera for capturing images obtained from the microscopy, and a microprocessor and software for controlling the system, wherein the method comprises the steps of: (a) dispensing the prepared sample into a receptacle of the light transparent sample plate, (b) obtaining a microscopic image of the sample without the LCD between the sample and the light source, (c) obtaining a microscopic image of the sample with the LCD oriented between the sample and the light source, (d) using the microprocessor and software to analyze and align the images obtained from (b) and (c) to obtain a composite image to locate and identify particles in the sample, and (e) using the result from (d) to instruct the microprocessor and software to selectively transmit or not transmit polymer-curing wavelengths of light to the curable polymeric medium at desired locations to provide an array of cured and uncured regions of the polymeric medium to selectively immobilize or not immobilize the particles of interest. ### Claim 2 A method for segregating particles from a sample prepared in a light-curable polymeric medium, utilizing a system, comprising: a light transparent sample plate having one or more receptacles for containing the sample, one or more light sources for illuminating and/or curing the sample, a liquid crystal display (LCD) for selectively controlling the transmission of light to the sample, a sample stage for aligning the sample plate and the LCD with respect to each other, an objective for observing the sample by microscopy, a camera for capturing images obtained from the microscopy, and a microprocessor and software for controlling the system, wherein the method comprises the steps of: (a) dispensing the prepared sample into a receptacle of the light transparent sample plate, (b) obtaining a microscopic image of the sample without the LCD between the sample and the light source, (c) obtaining a microscopic image of the sample with the LCD oriented between the sample and the light source, (d) using the microprocessor and software to analyze and align the images obtained from (b) and (c) to obtain a composite image to locate and identify particles in the sample, (e) using the result from (d) to instruct the microprocessor and software to selectively transmit or not transmit polymer-curing wavelengths of light to the curable polymeric medium at desired locations to provide an array of cured and uncured regions of the polymeric medium to selectively immobilize or not immobilize the particles of interest, and (f) removing the segregated particles from the sample by pipetting, washing, rinsing, or suction. ### Claim 3 A method for segregating particles from a sample prepared in a light-curable polymeric medium, comprising the steps of: (a) loading a sample in a photo-sensitive medium; (b) acquiring one or more images using one or more non-gelation lights; (c) positioning a display means between the sample and the one or more non-gelation lights; (d) displaying a pattern on to the display means; (e) acquiring a composite image using the one or more non-gelation lights; (f) calculating a location of one or more particles in the sample; (g) displaying a gelation pattern on to the display means; (h) forming at least one micro-hydrogel using gelation light; and (i) processing the sample by washing or elution. ### Claim 4 (depends on 3) The method of claim 3, wherein the display means is an LCD. ### Claim 5 A method for segregating particles from a sample prepared in a light-curable polymeric medium, comprising the steps of: (a) loading a sample in a photo-sensitive medium; (b) positioning a display means between the sample and the one or more non-gelation lights; (c) displaying a pattern on to the display means; (d) acquiring a composite image using the one or more non-gelation lights; (e) calculating a location of one or more particles in the sample; (f) displaying a gelation pattern on to the display means; (g) forming at least one micro-hydrogel using gelation light; and (h) processing the sample by washing or elution. ### Claim 6 (depends on 5) The method of claim 5, wherein the display means is an LCD. ### Claim 7 (depends on 1) The method according to claim 1 wherein said light transparent sample plate is a 96-well plate. ### Claim 8 (depends on 1) The method according to claim 1 wherein said LCD comprises a rectangular array of pixels. ### Claim 9 (depends on 8) The method according to claim 8 wherein each pixel of said LCD array comprises two or more sub-pixels. ### Claim 10 (depends on 8) The method according to claim 8 wherein each pixel of said LCD array comprises a green light emitting sub-pixel, a red light emitting sub-pixel, and a blue light emitting sub-pixel. ### Claim 11 (depends on 10) The method according to claim 10 wherein each pixel has a dimension of about 60 μm by about 60 μm and each subpixel has a dimension of about 20 μm by about 60 μm. ### Claim 12 (depends on 8) The method according to claim 8 wherein said rectangular array of pixels comprises a rectangular array of from about 500 to about 10,000 by about 500 to about 10,000 pixels. ### Claim 13 (depends on 12) The method according to claim 12 wherein said rectangular array of pixels comprises a rectangular array from about 1500 by about 2400 pixels. ### Claim 14 (depends on 1) The method according to claim 1 wherein said one or more light sources are selected from the group consisting of light emitting diode (LED) light sources, lasers, incandescent lights, fluorescent lights, ultraviolet lights, halogen lights, and xenon lights. ### Claim 15 (depends on 14) The method according to claim 14 wherein said LED light source emits white light. ### Claim 16 (depends on 15) The method according to claim 15 wherein said LED light source emits white light having a wavelength from about 390 nm to about 700 nm. ### Claim 17 (depends on 14) The method according to claim 14 wherein said LED light source emits blue/violet light having a wavelength from about 390 nm to about 490 nm. ### Claim 18 (depends on 14) The method according to claim 14 wherein said LED light source emits green light having a wavelength from about 520 nm to about 560 nm. ### Claim 19 (depends on 14) The method according to claim 14 wherein said LED light source emits red light having a wavelength from about 635 nm to about 700 nm. ### Claim 20 (depends on 14) The method according to claim 14 wherein said LED light source emits ultraviolet light having a wavelength from about 290 nm to about 390 nm. ### Claim 21 (depends on 1) The method according to claim 1 wherein said LCD is located under said sample plate, and said one or more light sources for illuminating and/or curing said sample are located under said LCD. ### Claim 22 (depends on 1) The method according to claim 1 wherein said objective for observing the sample by microscopy is located above said sample plate. ### Claim 23 (depends on 1) The method according to claim 1 wherein the sample is a biological sample. ### Claim 24 (depends on 23) The method according to claim 23 wherein the particles of the biological sample are selected from the group consisting of cells, biological particles, beads, extracellular material, and liquid droplets. ### Claim 25 (depends on 24) The method according to claim 24 wherein said cells are selected from the group consisting of circulating tumor cells (CTCs), blood cells, bacteria, primary cells, transformed cells, pathogenic cells, and rare immune cells. ### Claim 26 (depends on 1) The method according to claim 1 wherein said light curable polymeric medium is selected from a photo-reactive polymer that demonstrates an increase in viscosity when exposed to a light source having a wavelength from about 290 to about 400 nm. ### Claim 27 (depends on 26) The method according to claim 26 wherein said light curable polymeric medium is selected from gelatin derivatives, poly-ethylene glycol derivatives, alginate and its derivatives, collagen and its derivatives, Pluronic® and its derivatives, and mixtures thereof. ### Claim 28 (depends on 1) The method according to claim 1 wherein said one or more light sources are a light source combination for generating an optic array pattern. ### Claim 29 (depends on 28) The method according to claim 28 wherein said light source combination for generating the optic array pattern is selected from a light projector or an organic light-emitting diode (OLED).