# US 12,216,055 B2 · Microscope image capturing methods

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- Number: US 12,216,055 B2
- Document ID: US12216055B2
- Status: Granted
- Kind: B2
- Application number: 17/568,608
- Filed: 2022-01-04
- Published: 2022-06-30
- Granted: 2025-02-04
- Earliest claimed: 2017-01-26
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Adam Ezra Cohen; Vicente Jose Parot
- Web page: https://patentgrep.com/patent/US12216055B2

## CPC

- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G01N 21/6408: {with measurement of decay time, time resolved fluorescence}
- G02B 21/06: Means for illuminating specimens
- G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)}
- G02B 21/36: arranged for photographic purposes or projection purposes (G02B21/18 takes precedence){or digital imaging or video purposes including associated control and data processing arrangements (image data processing per se G06T)}
- G02B 21/365: {Control or image processing arrangements for digital or video microscopes (G02B21/361, G02B21/362 take precedence)}
- G06T 7/521: from laser ranging, e.g. using interferometry; from the projection of structured light
- G06T 7/80: Analysis of captured images to determine intrinsic or extrinsic camera parameters, i.e. camera calibration
- G06T 7/97: {Determining parameters from multiple pictures (depth or shape recovery from multiple images G06T7/55; stereo camera calibration G06T7/85)}
- G01N 2021/6419: {Excitation at two or more wavelengths}
- G01N 2201/0675: SLM
- G01N 2201/127: Calibration; base line adjustment; drift compensation
- G06T 2207/10056: Microscopic image
- 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
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL

## Abstract

The present disclosure relates to spatially modulating the light source used in microscopy. In some cases, a light source projects a sequence of two-dimensional spatial patterns onto a sample using a spatial light modulator. In some cases, the spatial patterns are based on Hadamard matrices. In some cases, an imaging device captures frames of image data in response to light emitted by the sample and orthogonal components of the image data are analyzed by cross-correlating the image data with the spatial pattern associated with each frame. A microscope may be calibrated by illuminating a sample with the sequence of spatial patterns, capturing image data, and storing calibration that maps each pixel of the spatial light modulator to at least one pixel of the imaging device.

## Claims

### Claim 1

A method, comprising:
illuminating, in a microscope, a sample with a sequence of two-dimensional spatial patterns using light of a first wavelength, wherein each two-dimensional spatial pattern of the sequence of two-dimensional spatial patterns is near orthogonal to the other two-dimensional spatial patterns of the sequence of two-dimensional spatial patterns, wherein a first two-dimensional spatial pattern is near orthogonal to a second two-dimensional spatial pattern if a correlation coefficient between the first two-dimensional spatial pattern and the second two-dimensional spatial pattern is less than or equal to 0.25;
capturing image data based on light of a second wavelength emitted by the sample in response to being illuminated by the light of the first wavelength, the image data comprising a plurality of frames, wherein each frame of the plurality of frames is associated with a respective two-dimensional spatial pattern of the sequence of two-dimensional spatial patterns; and
analyzing, using at least one processor, orthogonal components of each frame of the image data.

### Claim 2 (depends on 1)

The method of claim 1, wherein the sequence of two-dimensional spatial patterns is formed using a digital micro-mirror array.

### Claim 3 (depends on 1)

The method of claim 1, wherein each two-dimensional spatial pattern of the sequence of two-dimensional spatial patterns is configured such that neighboring locations within the sample are illuminated with orthogonal functions of intensity.

### Claim 4 (depends on 1)

The method of claim 1, wherein each two-dimensional spatial pattern of the sequence of two-dimensional spatial patterns is a binary spatial pattern.

### Claim 5 (depends on 1)

The method of claim 1, wherein each two-dimensional spatial pattern of the sequence of two-dimensional spatial patterns is based on a Hadamard matrix.

### Claim 6 (depends on 1)

The method of claim 1, further comprising analyzing the image data using calibration data that maps each pixel of a two-dimensional spatial light modulator that generates the sequence of two-dimensional spatial patterns to at least one pixel of an imaging device that captures the image data.