Super-resolution microscopy

Patent metadata

Field	Value
Publication number	US-10901230-B2
Application number	US-201716311652-A
Country	US
Kind code	B2
Filing date	Jun 20, 2017
Priority date	Jun 21, 2016
Publication date	Jan 26, 2021
Grant date	Jan 26, 2021

Abstract

Example super-resolution microscopy systems are described herein that are configured for relatively high throughput. The disclosed microscopy systems can be to generate an array of sub-diffraction activated areas for imaging. The microscopy systems can be to utilize imaging techniques that employ time delay integration to build up super-resolution images over time. The disclosed microscopy systems can utilize long-lived fluorophores in conjunction with wide field and patterned illumination to generate super-resolution images of a sample with relatively high throughput.

First claim

What is claimed is:

1. A super-resolution microscopy system, comprising:
an excitation light source to activate a fluorescent dye;
a depletion light source to photoswitch the fluorescent dye to an off-state;
an optical path comprising a wide field optic component for activating the fluorescent dye in a region using the excitation light source and an interference grid optic component that generates a pattern of light from the depletion light source that selectively deactivates fluorophores in the region while leaving targeted fluorophores in sub-diffraction areas active to form a patterned depletion grid;
a scanning system configured to move a sample;
one or more detectors that receive and integrate signals from the sub-diffraction areas over time and generate an integrated signal for the sub-diffraction areas; and
a processor programmed to determine fluorescence of the sub-diffraction areas from the integrated signal.

2. The system of claim 1 , wherein the sub-diffraction areas correspond to fluorescent nucleic acid molecules on a solid support.

3. The system of claim 1 , wherein the interference grid optic generates a standing wave grid from the depletion light source.

4. The system of claim 1 , wherein the one or more detectors comprise a detector for each sub-diffraction area.

5. The system of claim 1 , wherein the optical path includes a waveguide to generate a standing wave with the light from the depletion light source within the waveguide.

6. The system of claim 1 , wherein the one or more detectors comprises a single detector to detect light from the sample.

7. The system of claim 6 , wherein the single detector comprises a multi-channel photon detector.

8. The system of claim 7 , wherein the multi-channel photon detector comprises a CCD image sensor.

9. The system of claim 1 wherein the scanning system is configured to move the sample so that the sub-diffraction areas move relative to the sample.

10. The system of claim 1 , wherein the patterned depletion grid moves in synchronization with the sample moved by the scanning system in a first direction.

11. The system of claim 10 , wherein the patterned depletion grid remains stationary relative to the sample in a second direction.

12. The system of claim 1 , wherein the depletion light source is to photoswitch the fluorescent dye to an off-state for at least one second.

13. A super-resolution microscopy system, comprising:
an excitation light source to activate a fluorescent dye;
a depletion light source to photoswitch the fluorescent dye to an off-state;
an optical path comprising a wide field optic component for activating the fluorescent dye in a region using the excitation light source and an interference grid optic component that generates a plurality of patterned regions comprising a pattern of light from the depletion light source that selectively deactivates fluorophores while leaving targeted fluorophores in sub-diffraction areas active to form a patterned depletion grid;
one or more detectors configured to receive and integrate signals from fluorophores illuminated by the sub-diffraction areas and to generate an integrated signal for the sub-diffraction areas on the sample; and
a processor that receives the integrated signal from the one or more detectors and determines fluorescence of the fluorophores based on the integrated signal.

14. The system of claim 13 , wherein the fluorophores have a dark state with a lifetime that is greater than or equal to about 100 ms.

15. The system of claim 13 , wherein the off-state of the fluorescent dye is stable for at least 10 seconds.

16. The system of claim 15 , wherein the fluorescent dye comprises rhodamine, oxazine or carbocyanine dye.

17. A method of performing super-resolution microscopy to read a sample, the method comprising:
generating an array of sub-diffraction areas by (i) selectively activating fluorophores on a sample using an excitation light source and a wide field optic component and (ii) selectively de-activating fluorophores on the sample using a depletion light source and an interference grid optic component to selectively deactivate fluorophores of the sample leaving targeted fluorophores in the sub-diffraction areas active to form the patterned depletion grid;
receiving and integrating signals from the sub-diffraction areas over time using one or more detectors;
determining an integrated signal for the sub-diffraction areas on the sample; and
determining fluorescence of the sub-diffraction areas on the sample from the integrated signal.

18. The method of claim 17 , wherein the sample is an array of nucleic acid features on a solid support.

19. The method of claim 17 , wherein the one or more detectors comprises a detector for each sub-diffraction area in the array of sub-diffraction areas.

20. The method of claim 17 , wherein generating the array of sub-diffraction areas comprises generating a standing wave with the light from the depletion light.

21. The method of claim 17 , further comprising scanning the array of sub-diffraction areas across the sample in a first direction.

22. The method of claim 17 further comprising utilizing an imaging buffer that includes a reducing agent that reacts with a fluorescent dye of the fluorophores to chemically alter the fluorescent dye to a non-fluorescent, dark state.

23. The method of claim 22 further comprising introducing an oxidizing agent.

24. The method of claim 17 further comprising shifting the patterned depletion grid longitudinally or laterally.

25. A method of performing super-resolution microscopy to read a sample, the method comprising:
generating wide field activation illumination to excite fluorophores within an illuminated region;
generating patterned depletion illumination using an interference grid optic component to selectively photoswitch the fluorophores to an off-state in a targeted portion of the illuminated region while leaving targeted fluorophores in sub-diffraction areas active to form a patterned depletion grid;
receiving and integrating signals from the excited fluorophores within the illuminated region using one or more detectors;
generating an integrated signal for sub-diffraction areas on the sample; and
determining fluorescence of the sub-diffraction areas on the sample from the integrated signal.

26. The method of claim 25 , wherein the fluorophores have a dark state with a lifetime that is greater than or equal to about 100 ms.

27. The method of claim 25 , wherein the fluorophores comprise rhodamine, oxazine or carbocyanine dyes.

28. The method of claim 25 , further comprising moving the patterned depletion illumination using a scanning system so that the patterned depletion illumination is stationary with respect to the sample.

29. The method of claim 25 , further comprising associating signals generated by the one or more detectors with sub-diffraction areas on the sample using information from the scanning system such that an integrated signal for an sub-diffraction area on the sample is a result of selectively integrating the signals generated by the one or more detectors that received the light emitted from the sub-diffraction area on the sample.

30. The method of claim 25 , wherein the patterned depletion illumination generates regions of zero point intensity to selectively de-activate fl

Assignees

• Illumina Inc
• Illumina Cambridge Ltd

Inventors

• Skinner Gary Mark
• Evans Geraint Wyn
• Hong Stanley S
• Moon John A
• Bowen M Shane
• Boutell Jonathan Mark
• Betley Jason Richard

Classifications

• G02B21/0072

  details concerning resolution or correction, including general design of CSOM objectives · CPC title

• G02B21/0032

  Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers (G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06) · CPC title

• G02B27/58 Primary

  Optics for apodization or superresolution; Optical synthetic aperture systems · CPC title

• G02B21/0076

  arrangements using fluorescence or luminescence · CPC title

• G02B21/367

  providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison · CPC title