Multispectral sample imaging
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.
1. A method, comprising:
obtaining one or more images of a biological sample, wherein each sample image corresponds to a different combination of illumination light wavelengths and light emission wavelengths; and
obtaining a background image of the biological sample by exposing the biological sample to background excitation light and measuring light emission from the biological sample at one or more background emission wavelengths that are each within a wavelength range defined by the light emission wavelengths,
wherein for each of one or more spectral contributors in the biological sample exposed to the background excitation light, a spectral emission intensity at each of the background emission wavelengths is 10% or less of a maximum spectral emission intensity of the spectral contributor following excitation at each of the illumination light wavelengths; and
wherein a distribution of the background emission wavelengths has a full width at half maximum (FWHM) spectral width Δλ and center wavelength λ c , and the background image corresponds to measured light emission for at least some wavelengths within a range from λ c -λ/2 to λ c +λ/2.
2. A system, comprising:
a wavelength-adjustable light source; and
a detector,
wherein during operation of the system:
the light source is configured to generate illumination light in one or more illumination light bands and expose a biological sample to the illumination light;
the detector is configured to measure light emission from the biological sample to obtain N>1 sample images of the biological sample in response to the illumination light, wherein each sample image corresponds to a different combination of one or more of the illumination light bands and one or more wavelength bands of the light emission, and wherein the one or more wavelength bands of the light emission define a wavelength range;
the light source is configured to generate light in a background excitation band and expose the biological sample to the light in the background excitation band; and
the detector is configured to measure light emission from the biological sample in a background spectral band to obtain a background image of the biological 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 λ c within the wavelength range, and wherein at least some wavelengths within the background spectral band are within a range from λ c −λ/2 to λ c +λ/2; and
wherein for each of a plurality of spectral contributors in the sample exposed to the 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 spectral contributor following excitation of the spectral contributor in each of the illumination light bands and the background excitation band.
3. The system of claim 2 , further comprising a controller, wherein the controller is configured to obtain an autofluorescence image of the biological sample from the background image.
4. The system of claim 3 , wherein during operation of the system, the controller is configured to display the autofluorescence image on a display device.
5. The system of claim 3 , wherein during operation of the system, the controller is configured to determine, at each of multiple locations in the biological sample, an amount of autofluorescence emission from the biological sample.
6. The system of claim 5 , wherein during operation of the system, the controller is configured to adjust, for each of the multiple locations in the biological sample, and for one or more of the N sample images, values corresponding to sample emission intensity to correct for autofluorescence emission from the biological 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 biological sample.
7. The system of claim 6 , 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 in the biological sample.
8. The system of claim 7 , wherein during operation of the system, the controller is configured to:
decompose 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 biological sample.
9. The system of claim 8 , wherein during operation of the system, the controller is configured to decompose the at least some of the N sample images based on at least one pure spectrum of autofluorescence emission from the biological sample.
10. The system of claim 9 , 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.
11. The system of claim 3 , wherein during operation of the system, the controller is configured to classify pixels of one or more of the sample images into different classes based on information derived from the autofluorescence image.
12. The system of claim 11 , wherein the different classes correspond to different cell types in the biological sample.
13. The system of claim 2 , wherein for each of one or more non-endogenous spectral contributors in the sample exposed to 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 spectral emission intensity of the non-endogenous spectral contributor in each of illumination light bands and the background excitation band.
14. The system of claim 2 , wherein for each of one or more non-endogenous spectral contributors in the sample exposed to 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 spectral emission intensity of the non-endogenous spectral contributor in each of the illumination light bands and the background excitation band.
15. The system of claim 2 , wherein N>3.
16. The system of claim 2 , wherein N>5.
17. The system of claim 2 , wherein the biological sample comprises M non-endogenous spectral contributors, and wherein M≤N.
18. The system of claim 17 , wherein M>4.
19. The system of claim 18 , 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.
20. The system of claim 17 , wherein M>6.
21. The system of claim 17 , wherein the M non-endogenous spectral contributors comprise one or more fluorescent species that selectively bind to different chemical moieties in the biological sample.
22. The system of claim 21 , wherein the one or more fluorescent species comprise one or more immunofluorescent probes.
23. The system of claim 21 , wherein the M non-endogenous spectral contributors comprise one or more counterstains.
24. A computer-readable storage medium comprising instructions that, when executed by processing device, cause the processing device to:
obtain one or more images of a biological sample, wherein each sample image corresponds to a different combination of illumination light wavelengths and light emission wavelengths; and
obtain a background image of the biological sample by exposing the biological sample to background excitation light and measuring light emission from the biological sample at one or more background emission wavelengths that are each within a wavelength range defined by the light emission wavelengths,
wherein for each of one or more spectral contributors in the biological sample exposed to the background excitation light, a spectral emission intensity at each of the background emission wavelengths is 10% or less of a maximum spectral emission intensity of the spectral contributor following excitation at each of the illumination light wavelengths; and
wherein a distribution of the background emission wavelengths has a full width at half maximum (FWHM) spectral width Δλ and center wavelength λ c , and the background image corresponds to measured light emission for at least some wavelengths within a range from λ c −λ/2 to λ c +λ/2.