Enhancing visual assessment of samples
View Patent ↗Methods and systems for imaging a biological sample are disclosed, and include: (a) staining the sample with a first stain that includes eosin and at least one additional stain; (b) directing excitation light to the sample to cause each of the stains to emit fluorescence; and (c) recording images of the fluorescence emitted from the stains in the sample, where the amount of eosin in the sample is sufficiently dilute to cause the sample to have an average optical density of less than 0.10 at green wavelengths.
1. A method for imaging a biological sample, the method comprising:
a. staining the sample with a first stain comprising eosin and at least one additional stain;
b. directing excitation light to the sample to cause each of the stains to emit fluorescence; and
c. recording images of the fluorescence emitted from the stains in the sample,
wherein the amount of eosin in the sample is sufficiently dilute to cause the sample to have an average optical density of less than 0.10 at green wavelengths.
2. The method of claim 1 , wherein the amount of eosin in the sample is sufficiently dilute to cause the sample to have an average optical density of less than 0.05 at green wavelengths.
3. The method of claim 1 , wherein the staining of the sample with the first stain comprising eosin comprises contacting the sample for one second or less with a solution having an eosin concentration of 0.01 g or less of eosin per 100 mL of solution.
4. The method of claim 1 , wherein the amount of eosin in the sample is sufficient to produce detectable fluorescence by the eosin in response to the excitation light.
5. The method of claim 1 , wherein the green wavelengths are 530 nm through 570 nm.
6. The method of claim 1 , wherein the eosin comprises at least one of eosin Y, eosin B, and phloxine.
7. The method of claim 1 , wherein the recorded images are spectrally resolved into different spectral bands.
8. The method of claim 7 , wherein at least some of the spectral bands correspond to a fluorescence wavelength range for a respective one of the stains.
9. The method of claim 1 , wherein the biological sample comprises blood, cells, or tissue sections.
10. The method of claim 1 , wherein the at least one additional stain comprises a fluorescent nuclear counterstain.
11. The method of claim 10 , wherein the fluorescent counterstain comprises DAPI or Hoechst.
12. The method of claim 10 , wherein the at least one additional stain further comprises at least one immunofluorescent stain.
13. The method of claim 12 , wherein the immunofluorescent stain comprises quantum dots.
14. The method of claim 1 , wherein the directing of the excitation light comprises sequentially directing excitation light in different spectral bands to the sample.
15. The method of claim 1 , wherein the recording of the fluorescence comprises recording images of the fluorescence in each of multiple different spectral bands.
16. The method of claim 1 , further comprising computationally processing the recorded images to obtain information about the sample.
17. The method of claim 16 , wherein the processing of the recorded images comprises spectrally unmixing the recorded images into unmixed images, wherein each unmixed image corresponds to fluorescence from a respective one of the stains.
18. The method of claim 16 , wherein the processing of the recorded images comprises generating a bright-field image of the sample from the recorded fluorescence images.
19. The method of claim 18 , wherein the bright-field image comprises pink regions corresponding regions of the sample that localize eosin and blue regions corresponding to regions of the sample containing nuclei.
20. The method of claim 19 , wherein the bright-field image further comprises additional regions rendered to correspond to regions of the sample that localize an immunofluorescent stain.
21. The method of claim 16 , further comprising outputting the information about the sample.
22. The method of claim 21 , wherein the outputted information comprises a synthetic image derived from the recorded images.
23. The method of claim 1 , wherein a difference between the average optical density of the sample at the green wavelengths and an average optical density of the sample at red wavelengths is 0.08 or less.
24. The method of claim 1 , wherein the red wavelengths are 610 nm through 650 nm.
25. The method of claim 1 , wherein the contribution of the eosin to the average optical density of the sample is less than 0.05.
26. A method for imaging a biological sample, the method comprising:
a. staining the sample with a first stain comprising eosin, a nuclear counterstain, and at least one immunofluorescent stain, wherein the amount of eosin in the sample is sufficiently dilute to cause the sample to have an average optical density of less than 0.10 at green wavelengths;
b. directing excitation light to the sample to cause each of the stains to emit fluorescence, wherein the amount of eosin in the sample is sufficient to produce detectable fluorescence by the eosin in response to the excitation light;
c. recording images of the fluorescence emitted from the stains in the sample;
d. computationally processing the recorded images to produce a synthetic image; and
e. displaying the synthetic image.
27. The method of claim 26 , wherein the synthetic image comprises a bright-field image comprising pink regions corresponding regions of the sample that localize eosin, blue regions corresponding to regions of the sample containing nuclei, and additional regions rendered to correspond to regions of the sample that localize the immunofluorescent stain.
28. The method of claim 26 , wherein the processing of the recorded images comprises spectrally unmixing the recorded images into unmixed images, and wherein each unmixed image corresponds to fluorescence from a respective one of the stains.
29. The method of claim 26 , wherein the biological sample comprises blood.
30. The method of claim 26 , wherein the eosin in the sample has an average optical density of less than 0.05 at wavelengths between 530 nm and 580 nm.
31. The method of claim 26 , wherein a difference between the average optical density of the sample in the green wavelengths and an average optical density of the sample at red wavelengths is 0.08 or less.
32. The method of claim 26 , wherein the red wavelengths are 610 nm through 650 nm.
33. The method of claim 26 , wherein the contribution of the eosin to the average optical density of the sample is less than 0.05.
34. A method for imaging a biological sample, the method comprising:
a. staining the sample with a first stain comprising eosin and at least one additional stain;
b. measuring fluorescence emission from eosin and from the at least one additional stain in the sample; and
c. spectrally unmixing the measured fluorescence emission into contributions from eosin and from the at least one additional stain,
wherein the amount of eosin in the sample is sufficiently dilute to cause the sample to have an average optical density of less than 0.10 at green wavelengths.