# US 9,435,993 B2 · Three dimensional microscopy imaging > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,435,993 B2 - Document ID: US9435993B2 - Status: Granted - Kind: B2 - Application number: 14/224,039 - Filed: 2014-03-24 - Published: 2014-11-20 - Granted: 2016-09-06 - Earliest claimed: 2013-03-24 - Assignees: Bruker Nano, Inc. - Applicants: Vutara, Inc. - Inventors: Stan Kanarowski - Web page: https://patentgrep.com/patent/US9435993B2 ## CPC - 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} - G01N 21/64: Fluorescence; Phosphorescence - G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)} - G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)} - G02B 27/1066: {for enhancing image performance, like resolution, pixel numbers, dual magnifications or dynamic range, by tiling, slicing or overlapping fields of view} - G02B: OPTICAL ELEMENTS, SYSTEMS OR 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) ## Abstract A system and method for creating three dimensional images using probe molecules is disclosed and described. A sample is mounted on a stage. The sample has a plurality of probe molecules. The sample is illuminated with light, causing the probe molecules to luminesce. The probe luminescence can be split into at least four paths corresponding to at least four detection planes corresponding to object planes in the sample. The at least four detection planes are detected linearly via an sCMOS camera. Object planes in corresponding recorded regions of interest are recorded in the camera. A signal from the regions of interest is combined into a three dimensional image. ## Claims ### Claim 1 A method for creating three dimensional images using probe molecules, comprising: mounting a sample on a stage, the sample having a plurality of probe molecules; illuminating the sample with light to cause probe luminescence; splitting the probe luminescence into multiple paths corresponding one or more detection planes corresponding to object planes in the sample; detecting the one or more detection planes as a linear array via an sCMOS (scientific complementary metal-oxide-semiconductor) detector, wherein detecting the at least one or more detection planes via the sCMOS detector comprises detecting each detection plane in a different linear region of the sCMOS detector, and wherein the sCMOS detector comprises a rectangular sensor; recording the object planes in corresponding linear recording regions of interest of the sCMOS detector; and combining a signal from the regions of interest into a two or three dimensional image. ### Claim 2 (depends on 1) A method in accordance with claim 1, further comprising dichroically separating the probe luminescence into at least two wavelengths of light prior to or after splitting the probe luminescence, and wherein a first at least two of the multiple paths into which the probe luminescence is split correspond to a first wavelength of the at least two wavelengths, and a second at least two paths of the multiple paths into which the probe luminescence is split correspond to a second wavelength of the at least two wavelengths. ### Claim 3 (depends on 1) A method in accordance with claim 1, wherein illuminating the sample with light further comprises illuminating the sample with an activation light to activate at least one subset of the plurality of probe molecules, and subsequently illuminating the sample with an excitation light to cause probe luminescence. ### Claim 4 (depends on 1) A method in accordance with claim 1, further comprising separately recording the object planes for different regions of the sample and arranging the recording planes for the different regions together as a composite two or three dimensional image based on a different-wave length weighted calibration offset. ### Claim 5 (depends on 1) A method in accordance with claim 1, further comprising blurring the light using a blurring device between a light source and the stage. ### Claim 6 (depends on 5) A method in accordance with claim 5, wherein the blurring device comprises a vibration motor configured to vibrate and cause movement of a light guide to disrupt the light passing through the light guide. ### Claim 7 (depends on 1) A method in accordance with claim 1, further comprising forming a consistent light source by shaping the light source. ### Claim 8 (depends on 7) A method in accordance with claim 7, wherein the shaping is accomplished using a set of locked resonant scanners running a different speeds. ### Claim 9 (depends on 7) A method in accordance with claim 7, wherein the shaping is accomplished using microelectromechanical systems (MEMS) mirrors. ### Claim 10 (depends on 4) A method in accordance with claim 4, wherein the blurring device comprises an astigmatic lens or a photomask oriented between the between an objective and the sCMOS camera to enable point spread function engineering. ### Claim 11 (depends on 1) A method in accordance with claim 1, further comprising combining a two class calibration to create improved imaging data. ### Claim 12 (depends on 11) The method in accordance with claim 11, wherein the two class calibration corresponds to a first class of parameters specific to the sensor used in the detector and a second class of parameters related to typical imaging conditions. ### Claim 13 (depends on 11) The method claim 11, wherein classes of parameters in the two class calibration are determined based on characteristics of a microscopy system for performing the method on a onetime or regular basis. ### Claim 14 (depends on 11) The method of claim 11, wherein either class of the two class calibration is processed as part of post processing at or near the sCMOS detector before being transferred to a computer. ### Claim 15 (depends on 14) The method of claim 14, wherein the processing near the detector is performed by a processor embedded in an integrated circuit. ### Claim 16 (depends on 14) The method of claim 14, wherein the imaging data with irrelevant to the microscopy imaging is removed through pre-filtering prior to being transferred to a computer. ### Claim 17 (depends on 14) The method of claim 14, wherein the imaging data is adjusted to compensate for effects of a rolling shutter of the sCMOS detector. ### Claim 18 (depends on 11) The method of claim 11, wherein the two class calibration adjusts for wavelength of light or light intensity or length of per frame imaging time. ### Claim 19 (depends on 11) The method of claim 11, wherein the two class calibration adjusts for sample and imaging conditions of ambient light or embedding medium or cover slip or temperature. ### Claim 20 (depends on 11) The method of claim 11, wherein the two class calibration is dependent on the behavior of pixels within a neighborhood. ### Claim 21 (depends on 1) A method in accordance with claim 1, further comprising operating the sCMOS detector in a rolling shutter mode and compensating for a time delay resulting from rolling of a shutter of the sCMOS detector. ### Claim 22 A method for creating three dimensional images using probe molecules, comprising: mounting a sample on a stage, the sample having a plurality of probe molecules; illuminating the sample with light to cause probe luminescence; splitting the probe luminescence into multiple paths corresponding one or more detection planes corresponding to object planes in the sample; detecting the one or more detection planes as a linear array via an sCMOS (scientific complementary metal-oxide-semiconductor) detector; recording the object planes in corresponding linear recording regions of interest of the sCMOS detector; and combining a signal from the regions of interest into a two or three dimensional image, wherein the recording includes separately recording the object planes for different regions of the sample and the combining includes arranging the recording planes for the different regions together as a composite two or three dimensional image based on a different-wave length weighted calibration offset. ### Claim 23 (depends on 22) A method in accordance with claim 22, wherein detecting the at least one or more detection planes via the sCMOS detector comprises detecting each detection plane in a different linear region of the sCMOS detector, and wherein the sCMOS detector comprises a rectangular sensor. ### Claim 24 (depends on 22) A method in accordance with claim 22, further comprising blurring the light using a blurring device between a light source and the stage. ### Claim 25 (depends on 22) A method in accordance with claim 22, further comprising combining a two class calibration to create improved imaging data. ### Claim 26 (depends on 22) A method in accordance with claim 22, further comprising operating the sCMOS detector in a rolling shutter mode and compensating for a time delay resulting from rolling of a shutter of the sCMOS detector. ### Claim 27 (depends on 1) A method in accordance with claim 1, wherein illuminating the sample with light further comprises illuminating the sample with an activation light to activate at least one subset of the plurality of probe molecules, and subsequently illuminating the sample with an excitation light to cause probe luminescence. ### Claim 28 A method for creating three dimensional images using probe molecules, comprising: mounting a sample on a stage, the sample having a plurality of probe molecules; illuminating the sample with light to cause probe luminescence; splitting the probe luminescence into multiple paths corresponding one or more detection planes corresponding to object planes in the sample; detecting the one or more detection planes as a linear array via an sCMOS (scientific complementary metal-oxide-semiconductor) detector; recording the object planes in corresponding linear recording regions of interest of the sCMOS detector; and combining a signal from the regions of interest into a two or three dimensional image, wherein the combining includes a two class calibration to create improved imaging data, wherein either class of the two class calibration is processed as part of post processing at or near the sCMOS detector before being transferred to a computer. ### Claim 29 (depends on 28) A method in accordance with claim 28, wherein illuminating the sample with light further comprises illuminating the sample with an activation light to activate at least one subset of the plurality of probe molecules, and subsequently illuminating the sample with an excitation light to cause probe luminescence. ### Claim 30 (depends on 28) A method in accordance with claim 28, further comprising separately recording the object planes for different regions of the sample and arranging the recording planes for the different regions together as a composite two or three dimensional image based on a different-wave length weighted calibration offset. ### Claim 31 (depends on 28) A method in accordance with claim 28, further comprising blurring the light using a blurring device between a light source and the stage.