# US 11,442,260 B2 · Imaging a sample in a sample holder > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,442,260 B2 - Document ID: US11442260B2 - Status: Granted - Kind: B2 - Application number: 16/041,922 - Filed: 2018-07-23 - Published: 2019-02-14 - Granted: 2022-09-13 - Earliest claimed: 2017-08-11 - Assignees: Tecan Trading AG - Applicants: Tecan Trading AG - Inventors: Andreas Erlbacher; Markus Schürf; Josef Grassl; Alexander Kaser - Web page: https://patentgrep.com/patent/US11442260B2 ## CPC - G02B 21/006: {focusing arrangements; selection of the plane to be imaged} - G02B 7/32: using active means, e.g. light emitter {(including both an active and a passive focus detecting device G02B7/285; using ultrasound G02B7/40)} - G02B 7/36: using image sharpness techniques {, e.g. image processing techniques for generating autofocus signals (image data processing per se G06T; in cameras having a solid state image sensor H04N23/67)} - G02B 21/06: Means for illuminating specimens - G02B 21/244: {using image analysis techniques} - G02B 21/245: {using auxiliary sources, detectors} - G02B 21/365: {Control or image processing arrangements for digital or video microscopes (G02B21/361, G02B21/362 take precedence)} - G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS ## Abstract A system 100 and method are provided for imaging a sample in a sample holder. For providing autofocus, a 2D pattern is projected onto the sample holder 050 via an astigmatic optical element 120. Image data 172 of the sample is acquired by an image sensor 140 via magnification optics 150. A difference in sharpness of the two-dimensional pattern in the image data is measured along a first axis and a second axis. Based on the difference, a magnitude and direction of defocus of the camera subsystem is determined with respect to the sample holder. This enables the sample holder, and thereby the sample, to be brought into focus in a fast and reliable manner. ## Claims ### Claim 1 An imaging system for imaging a sample in a sample holder, the imaging system comprising: a camera subsystem comprising an image sensor and magnification optics and configured to image the sample via the magnification optics; a projection subsystem comprising a light source and an astigmatic optical element and configured to project a two-dimensional pattern onto the sample holder via the astigmatic optical element and the magnification optics; an actuator configured to adjust a distance between the sample holder and the magnification optics; and a processor subsystem comprising: a camera interface configured to receive image data acquired by the camera subsystem; an actuator interface configured to control the actuator; a memory comprising instruction data representing a set of instructions; and a processor configured to communicate with the camera interface and the memory and to execute the set of instructions, wherein execution of the set of instructions by the processor causes the processor to: determine a measure of sharpness of the two-dimensional pattern along a first axis in the image data; determine a measure of sharpness of the two-dimensional pattern along a second axis in the image data which is orthogonal to the first axis; determine a difference between the measures of sharpness along the first axis and the second axis, and based on the difference, determine a magnitude and direction of defocus of the camera subsystem with respect to the sample holder; and based on the magnitude and/or direction of defocus, control the actuator via the actuator interface to adjust the distance between the sample holder and the magnification optics. ### Claim 2 (depends on 1) The imaging system according to claim 1, wherein execution of the set of instructions by the processor causes the processor to determine each measure of sharpness on the basis of a spatial filtering operation applied to the image data, or a frequency transform of the image data. ### Claim 3 (depends on 1) The imaging system according to claim 1, wherein the astigmatic optical element is a tilted glass plate or a cylindrical lens. ### Claim 4 (depends on 1) The imaging system according to claim 1, wherein the projection subsystem further comprises an optical element configured to collimate light emitted by the light source after passing through the astigmatic optical element. ### Claim 5 (depends on 1) The imaging system according to claim 1, further comprising a mirror configured to, with one side, reflect at least part of light which is reflected by the sample and which has passed through the magnification optics onto the image sensor, and with the other side, transmit at least part of the light which is emitted by the light source towards the magnification optics. ### Claim 6 (depends on 1) The imaging system according to claim 1, wherein the two-dimensional pattern is a two-dimensional grid. ### Claim 7 (depends on 6) The imaging system according to claim 6, wherein the two-dimensional grid has a number of horizontal grid lines and a number of vertical grid lines each in the range of 5 to 15 grid lines. ### Claim 8 (depends on 1) The imaging system according to claim 1, wherein the two-dimensional pattern is provided by a plate which is arranged between the light source and the astigmatic optical element. ### Claim 9 (depends on 1) The imaging system according to claim 1, wherein execution of the set of instructions by the processes, causes the processor to, based on the magnitude and direction of defocus, control the actuator via the actuator interface to bring the sample or the sample holder into focus. ### Claim 10 (depends on 1) The imaging system according to claim 1, wherein the light source is a light emitting diode. ### Claim 11 (depends on 1) The imaging system according to claim 1, wherein the imaging system is a microplate reader. ### Claim 12 (depends on 1) The imaging system according to claim 1, wherein the sample holder is a well of a microplate or a slide. ### Claim 13 (depends on 1) A non-transitory computer-readable medium comprising a computer program, the computer program comprising instructions for causing a processor subsystem of the imaging system of claim 1 to perform a method comprising: receiving image data acquired by the camera subsystem; determining a measure of sharpness of the two-dimensional pattern along a first axis in the image data; determining a measure of sharpness of the two-dimensional pattern along a second axis in the image data which is orthogonal to the first axis; determining a difference between the measures of sharpness along the first axis and the second axis, and based on the difference, determine a magnitude and direction of defocus of the camera subsystem with respect to the sample holder; and based on the magnitude and/or direction of defocus, controlling the actuator to adjust the distance between the sample holder and the magnification optics. ### Claim 14 A computer-implemented method of imaging a sample in a sample holder using an imaging system, the imaging system comprising: a camera subsystem comprising an image sensor and magnification optics and configured to image the sample via the magnification optics; a projection subsystem comprising a light source and an astigmatic optical element and configured to project a two-dimensional pattern onto the sample holder via the astigmatic optical element and the magnification optics; an actuator configured to adjust a distance between the sample holder and the magnification optics; the method, performed by a processor of the imaging system, comprising: receiving image data acquired by the camera subsystem; determining a measure of sharpness of the two-dimensional pattern along a first axis in the image data; determining a measure of sharpness of the two-dimensional pattern along a second axis in the image data which is orthogonal to the first axis; determining a difference between the measures of sharpness along the first axis and the second axis, and based on the difference, determine a magnitude and direction of defocus of the camera subsystem with respect to the sample holder; and based on the magnitude and/or direction of defocus, controlling the actuator to adjust the distance between the sample holder and the magnification optics.