# US 12,111,456 B2 · Methods and apparatus for optimised interferometric scattering microscopy > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,111,456 B2 - Document ID: US12111456B2 - Status: Granted - Kind: B2 - Application number: 17/767,274 - Filed: 2020-10-09 - Published: 2022-11-17 - Granted: 2024-10-08 - Earliest claimed: 2019-10-10 - Assignees: Refeyn Ltd; Oxford University Innovation Limited - Applicants: Refeyn Ltd; Oxford University Innovation Limited - Inventors: Matthias Karl Franz Langhorst; Daniel Richard Cole; David John Lehar Graham; Philipp Kukura; Lee Priest - Web page: https://patentgrep.com/patent/US12111456B2 ## CPC - G02B 21/14: affording illumination for phase-contrast observation - G02B 21/18: Arrangements with more than one light path, e.g. for comparing two specimens - G02B 21/361: {Optical details, e.g. image relay to the camera or image sensor (G02B21/364 takes precedence; illumination details G02B21/06 and subgroups)} - 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} - H04N 23/72: Combination of two or more compensation controls - G02B 21/0092: {Polarisation microscopes} - G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS - H04N: PICTORIAL COMMUNICATION, e.g. TELEVISION ## Abstract The application discloses a method and apparatus for imaging a sample by interferometric scattering microscopy, the method comprising illuminating a sample with at least one coherent light source, the sample being held at a sample location comprising an interface having a refractive index change, illuminating the sample with illuminating radiation to generate a backpropagating signal from the sample comprising light reflected at the interface and light scattered by the sample, splitting the backpropagating signal into first and second signals, modifying the second signal using a modifying element such that the second signal differs from the first signal, directing the first and second signals onto first and second detectors to generate, respectively, first and second images and comparing, by a processor, the first and second images to determine one or more characteristics of the sample. ## Claims ### Claim 1 A method of imaging a sample by interferometric scattering microscopy, the method comprising: illuminating a sample with at least one coherent light source, the sample being held at a sample location comprising an interface having a refractive index change, illuminating the sample with illuminating radiation to generate a backpropagating signal from the sample comprising light reflected at the interface and light scattered by the sample; splitting the backpropagating signal into first and second signals; modifying the second signal using a modifying element such that the second signal differs from the first signal; directing the first and second signals onto first and second detectors to generate, respectively, first and second images; and comparing, by a processor, the first and second images to determine one or more characteristics of the sample. ### Claim 2 (depends on 1) The method according to claim 1 further comprising passing at least one of the first and the second signals through a spatial filter, the spatial filter being configured to effect a reduction in intensity on incident radiation, the reduction in intensity being greater within a predetermined numerical aperture. ### Claim 3 (depends on 1) The method of claim 1, wherein the at least one coherent light source comprises a first laser and a second laser, the beams of which are combined prior to illumination of the sample. ### Claim 4 (depends on 1) The method of claim 1, wherein splitting the signal comprises splitting the backpropagating signal into two signals having orthogonal polarizations. ### Claim 5 (depends on 1) The method of claim 1, wherein splitting the signal comprises splitting the backpropagating signal into two signals having different optical power. ### Claim 6 (depends on 5) The method of claim 5, wherein passing at least one of the first and second signals through a spatial filter comprises passing the first signal through a first spatial filter and modifying the second signal such that it differs from the first signal comprises passing the second signal through a second spatial filter wherein the first spatial filter and the second spatial filter are each configured to effect a reduction in intensity on incident radiation, the reduction in intensity being greater within a predetermined numerical aperture, wherein the first signal has a greater optical power than the second signal and wherein the second spatial filter applies a greater reduction in intensity to incident radiation than the first spatial filter. ### Claim 7 (depends on 1) The method of claim 1, wherein modifying the second signal comprises adjusting the phase of the second signal with respect to the first signal. ### Claim 8 (depends on 7) The method of claim 7, wherein adjusting the phase of the second signal with respect to the first signal comprises passing the second signal through a phase shift mask. ### Claim 9 (depends on 7) The method of either claim 7, wherein adjusting the second signal comprises passing the second signal through an imaging lens at an appropriate location along the optical path of the first signal. ### Claim 10 (depends on 7) The method of claim 7, wherein adjusting the phase of the second signal with respect to the first signal comprises adjusting the phase of the second signal with respect to the first signal by half a wavelength of the illuminating radiation. ### Claim 11 (depends on 1) The method of claim 1, wherein modifying the second signal comprises: passing the second signal through an optical element configured to apply asymmetric magnification to the second signal along a first dimension corresponding to an x-dimension of a pixel grid of the second detector. ### Claim 12 (depends on 11) The method of claim 11, wherein the method further comprises: and passing the first signal through an optical element configured to apply asymmetric magnification to the first signal along a second dimension, the second direction being orthogonal to the first direction and corresponding to a y-dimension of a pixel grid of the second detector. ### Claim 13 (depends on 1) The method of claim 1, wherein the at least one coherent light source is configured to provide illuminating light having at least two distinct interrogating wavelengths; and wherein splitting the signal comprises splitting the signal by wavelength, such that light having a first interrogating wavelength is directed to the first detector and light having a second interrogating wavelength is directed to the second detector. ### Claim 14 (depends on 1) The method of claim 1, wherein the predetermined numerical aperture is identical or similar to the numerical aperture of the illuminating light reflected from the sample location. ### Claim 15 (depends on 1) The method of claim 1, wherein modifying the second signal comprises phase shifting, magnifying in a first direction or spatial filtering with a greater intensity than the spatial filtering applied to the first signal. ### Claim 16 (depends on 1) An interferometric scattering microscope configured to carry out the method of claim 1, comprising: a sample location comprising a reflective surface; at least one coherent light source configured to illuminate the sample location; first and second detectors; a beam splitter configured to split a backpropagating signal from the sample location into first and second signals; and a modifying element configured to modify the second signal such that it differs from the first signal; wherein the system is configured to direct the first and second signals onto the first and second detectors, respectively. ### Claim 17 (depends on 16) An interferometric scattering microscope according to claim 16 further comprising at least one spatial filter positioned to filter at least one of the first signal and the second signal, wherein the spatial filter is configured affect a reduction in intensity on incident radiation that is greater within a predetermined numerical aperture.