# US 11,307,142 B2 · Multispectral sample imaging

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- Number: US 11,307,142 B2
- Document ID: US11307142B2
- Status: Granted
- Kind: B2
- Application number: 16/403,401
- Filed: 2019-05-03
- Published: 2019-11-07
- Granted: 2022-04-19
- Earliest claimed: 2018-05-03
- Assignees: Akoya Biosciences, Inc.
- Applicants: Akoya Biosciences, Inc.
- Inventors: Peter J. Miller; Kent S. Johnson; Carla Coltharp
- Web page: https://patentgrep.com/patent/US11307142B2

## CPC

- G01N 21/6456: {Spatial resolved fluorescence measurements; Imaging}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G01J 3/0297: {Constructional arrangements for removing other types of optical noise or for performing calibration}
- G01J 3/28: Investigating the spectrum (using colour filters G01J3/51)
- G01J 3/2823: {Imaging spectrometer}
- G01J 3/4406: {Fluorescence spectrometry}
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G06T 7/0012: {Biomedical image inspection}
- G06T 7/194: involving foreground-background segmentation
- G01J 2003/2826: {Multispectral imaging, e.g. filter imaging}
- G01N 2021/6417: {Spectrofluorimetric devices}
- G01N 2021/6419: {Excitation at two or more wavelengths}
- G01N 2021/6421: {Measuring at two or more wavelengths}
- G06T 2207/10036: Multispectral image; Hyperspectral image
- G06T 2207/10056: Microscopic image
- G06T 2207/10064: Fluorescence image
- G06T 2207/30024: Cell structures in vitro; Tissue sections in vitro
- 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)
- G01J: MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY (light sources F21, H01J, H01K, H05B; investigating properties of materials by optical means G01N)
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL

## Abstract

The disclosure features methods that include exposing a biological sample to illumination light and measuring light emission from the sample to obtain N sample images, where each sample image corresponds to a different combination of a wavelength band of the illumination light and one or more wavelength bands of the light emission, where the one or more wavelength bands of the light emission define a wavelength range, and where N>1, and exposing the sample to illumination light in a background excitation band and measuring light emission from the sample in a background spectral band to obtain a background image of the sample, where the background spectral band corresponds to a wavelength within the wavelength range.

## Claims

### Claim 1

A method, comprising:
exposing a plurality of non-endogenous spectral contributors in a biological sample to illumination light and measuring light emission from the sample to obtain N sample images, wherein each sample image corresponds to a different combination of a wavelength band of the illumination light and one or more wavelength bands of the light emission, wherein multiple wavelength bands of the light emission from the plurality of non-endogenous spectral contributors define a wavelength range, and wherein N>1; and
exposing the sample to illumination light in a background excitation band and measuring light emission from the sample in a background spectral band to obtain a background image of the sample, wherein the background spectral band comprises a distribution of wavelengths having a full width at half maximum (FWHM) spectral width Δλ and a center wavelength λc within the wavelength range, and wherein the wavelengths within the background spectral band correspond to wavelengths within a range from λc−Δλ/2 to λc+Δλ/2,
wherein for each of the plurality of non-endogenous spectral contributors in the sample exposed to the illumination light in the background excitation band, a spectral emission intensity at each wavelength within the background spectral band is 10% or less of a maximum measured spectral emission intensity of the non-endogenous spectral contributor following excitation of the non-endogenous spectral contributor in each of the wavelength bands of the illumination light and the background excitation band.

### Claim 2 (depends on 1)

The method of claim 1, further comprising obtaining an autofluorescence image of the sample from the background image.

### Claim 3 (depends on 2)

The method of claim 2, further comprising displaying the autofluorescence image on a display device.

### Claim 4 (depends on 2)

The method of claim 2, further comprising determining, at each of multiple locations in the sample, an amount of autofluorescence emission from the sample.

### Claim 5 (depends on 4)

The method of claim 4, further comprising, at each of the multiple locations in the sample, and for one or more of the N sample images:
adjusting values corresponding to sample emission intensity to correct for autofluorescence emission from the sample based on the amount of autofluorescence emission at each of the multiple locations and at least one pure spectrum of autofluorescence emission from the sample.

### Claim 6 (depends on 5)

The method of claim 5, wherein the at least one pure spectrum of autofluorescence emission comprises multiple pure spectra of autofluorescence emission, and wherein the multiple pure spectra of autofluorescence emission each correspond to a different subset of the multiple locations.

### Claim 7 (depends on 6)

The method of claim 6, further comprising:
decomposing at least some of the N sample images based on the amount of autofluorescence emission from the sample at each of the multiple locations to obtain M spectral contributor images, wherein each of the M spectral contributor images corresponds to light emission only from a different one of the non-endogenous spectral contributors; and
at each of the multiple locations, determining an amount of the M non-endogenous spectral contributors in the sample.

### Claim 8 (depends on 7)

The method of claim 7, further comprising decomposing the at least some of the N sample images based on at least one pure spectrum of autofluorescence emission from the sample.

### Claim 9 (depends on 8)

The method of claim 8, wherein the at least one pure spectrum of autofluorescence emission comprises multiple pure spectra of autofluorescence emission, and wherein the multiple pure spectra of autofluorescence emission each correspond to a different subset of the multiple locations.

### Claim 10 (depends on 2)

The method of claim 2, further comprising classifying pixels of one or more of the sample images into different classes based on information derived from the autofluorescence image.

### Claim 11 (depends on 10)

The method of claim 10, wherein the different classes correspond to different cell types in the sample.

### Claim 12 (depends on 1)

The method of claim 1, wherein for each of the plurality of non-endogenous spectral contributors in the sample exposed to the illumination light in the background excitation band, the spectral emission intensity at each wavelength within the background spectral band is 4% or less of the maximum measured spectral emission intensity of the non-endogenous spectral contributor following excitation of the sample in each of the wavelength bands of the illumination light and the background excitation band.

### Claim 13 (depends on 1)

The method of claim 1, wherein for each of the plurality of non-endogenous spectral contributors in the sample exposed to the illumination light in the background excitation band, the spectral emission intensity at each wavelength within the background spectral band is 2% or less of the maximum measured spectral emission intensity of the non-endogenous spectral contributor following excitation of the sample in each of the wavelength bands of the illumination light and the background excitation band.

### Claim 14 (depends on 1)

The method of claim 1, wherein N>3.

### Claim 15 (depends on 1)

The method of claim 1, wherein N>5.

### Claim 16 (depends on 1)

The method of claim 1, wherein the sample comprises M non-endogenous spectral contributors, and wherein M≤N.

### Claim 17 (depends on 16)

The method of claim 16, wherein M>4.

### Claim 18 (depends on 17)

The method of claim 17, wherein a sum of spectral emission intensities of each non-endogenous spectral contributor in the sample at each wavelength within the background spectral band is 10% or less of a total fluorescence emission intensity in the background spectral band.

### Claim 19 (depends on 16)

The method of claim 16, wherein M>6.

### Claim 20 (depends on 16)

The method of claim 16, wherein the M non-endogenous spectral contributors comprise one or more fluorescent species that selectively bind to different chemical moieties in the sample.

### Claim 21 (depends on 20)

The method of claim 20, wherein the one or more fluorescent species comprise one or more immunofluorescent probes.

### Claim 22 (depends on 20)

The method of claim 20, wherein the M non-endogenous spectral contributors comprise one or more counterstains.