Rapid adaptive optical microscopy over large multicellular volumes

Patent metadata

Field	Value
Publication number	US-9500846-B2
Application number	US-201514660906-A
Country	US
Kind code	B2
Filing date	Mar 17, 2015
Priority date	Mar 17, 2014
Publication date	Nov 22, 2016
Grant date	Nov 22, 2016

Abstract

Excitation light is focused to a focus within a sample and the focus is scanned within a volume in the sample with scanning optical elements. Signal light emitted from the focus is de-scanned, with the one or more scanning optical elements, onto a wavefront sensor as the focus is scanned within the volume. Based on the descanned signal light, an average aberration created by the volume of the sample of a wavefront of the excitation light is determined. A wavefront of the excitation light is corrected by an amount according to the determined average aberration while the focus is scanned within the volume, the signal light is imaged onto a photosensitive detector as the focus is scanned within the volume, and a wavefront of the imaged signal light is corrected by an amount according to the determined average aberration while the focus is scanned. These steps can be repeated for a plurality of different volumes in the sample, and an image of the sample can be generated based on the detected signal light from scanned foci within the different volumes.

First claim

What is claimed is:

1. A method comprising:
(a) focusing first excitation light having a first wavelength to a first focus within a sample;
(b) scanning the first focus within a volume in the sample with one or more scanning optical elements;
(c) de-scanning, with the one or more scanning optical elements, as the first focus is scanned within the volume, first signal light emitted from the first focus onto a wavefront sensor;
(d) determining, based on the first signal light that is descanned onto the wavefront sensor, an average aberration created by the volume of the sample of a wavefront of the first excitation light;
(e) focusing second excitation light having a second wavelength through the objective lens to a second focus within the volume in the sample;
(e) scanning the second focus within the volume in the sample with the one or more scanning optical elements;
(f) correcting a wavefront of the second excitation light by an amount according to the determined average aberration while the second focus is scanned within the volume;
(g) imaging second signal light emitted from the sample in response to the second excitation light onto a photosensitive detector as the second focus is scanned within the volume,
(h) correcting a wavefront of the second signal light by an amount according to the determined average aberration while the second focus is scanned within the volume;
(i) repeating (a)-(h) for a plurality of different volumes in the sample; and
(j) generating an image of the sample based on the detected second signal light from scanned foci within the different volumes.

2. The method of claim 1 , wherein the first wavelength is longer than a wavelength of the first signal light and wherein the first wavelength is longer than the second wavelength.

3. The method of claim 1 , wherein an optical path between the sample and the photosensitive detector includes a pinhole aperture in an opaque screen, wherein the pinhole is optically conjugate to the volume in the sample.

4. The method of claim 1 , wherein correcting the wavefront of the second excitation light includes modulating a beam of the second excitation light by a wavefront modulating element and controlling portions of the wavefront modulating element to impart changes to subportions of the beam to achieve the correction of the wavefront of the second excitation light, and wherein the scanning optical elements and the wavefront modulating element are all optically conjugate to a rear pupil of the objective lens.

5. The method of claim 1 , wherein correcting the wavefront of the second excitation light includes reducing a spatial extent of the second focus.

6. The method of claim 1 , wherein the first wavelength is greater than 800 nm and wherein a wavelength of the first signal light is less than 800 nm.

7. The method of claim 1 , wherein the first signal light emitted from the volume includes light emitted from a volume substantially centered at the first focus and wherein the volume has a spatial extent transverse to the optical axis of the objective lens that is greater than the diffraction-limited resolution of the objective lens.

8. The method of claim 1 , wherein determining the average aberration created by the volume of the sample includes determining the aberration based on first signal light that is de-scanned onto the first photosensitive sensor from the volume within the sample that is greater than 2 μm 3 .

9. The method of claim 1 , wherein the wavefront sensor includes a direct wavefront sensor.

10. The method of claim 1 , wherein the wavefront sensor includes a Shack-Hartmann sensor.

11. A method comprising:
(a) focusing excitation light to a focus within a sample;
(b) scanning the focus within a volume in the sample with one or more scanning optical elements;
(c) de-scanning, with the one or more scanning optical elements, as the focus is scanned within the volume, signal light emitted from the focus onto a wavefront sensor;
(d) determining, based on the signal light that is descanned onto the wavefront sensor, an average aberration created by the volume of the sample of a wavefront of the excitation light;
(e) correcting a wavefront of the excitation light by an amount according to the determined average aberration while the focus is scanned within the volume;
(f) imaging the signal light onto a photosensitive detector as the focus is scanned within the volume;
(g) correcting a wavefront of the imaged signal light by an amount according to the determined average aberration while the focus is scanned within the volume;
(h) repeating (a)-(g) for a plurality of different volumes in the sample; and
(j) generating an image of the sample based on the imaged signal light from scanned foci within the different volumes.

12. The method of claim 11 ,
wherein the determined average aberration is determined for a first subportion of the volume that is scanned, and
wherein the corrected wavefront of the excitation light and the corrected wavefront of the imaged signal light are corrected based on the determined average aberration for the first subportion, when the determined focus is scanned over a second subportion of the volume.

13. The method of claim 12 ,
wherein the focus is scanned over the first subportion and over the second subportion sequentially.

14. The method of claim 13 , wherein the focus is scanned sequentially over at least five subportions, including the first and second subportions.

15. The method of claim 11 ,
wherein a wavelength of the excitation light is approximately twice as great as a wavelength of the signal light.

16. The method of claim 11 , wherein correcting the wavefront of the excitation light includes modulating a beam of the excitation light by a wavefront modulating element and controlling portions of the wavefront modulating element to impart changes to subportions of the beam to achieve the correction of the wavefront of the excitation light, and wherein the scanning optical elements and the wavefront modulating element are all optically conjugate to a rear pupil of the objective lens.

17. The method of claim 11 , wherein correcting the wavefront of the excitation light includes reducing a spatial extent of the second focus.

18. The method of claim 11 , wherein the wavelength of the excitation light is greater than 800 nm and wherein a wavelength of the signal light is less than 800 nm.

19. The method of claim 11 , wherein the signal light emitted from the volume has a spatial extent transverse to the optical axis of the objective lens that is greater than the diffraction-limited resolution of the objective lens.

20. The method of claim 11 , wherein determining the average aberration created by the volume of the sample includes determining the aberration based on signal light that is de-scanned onto the wavefront sensor from the volume within the sample that is greater than 2 μm 3 .

21. The method of claim 11 , wherein the wavefront sensor includes a direct wavefront sensor.

22. The method of claim 11 , wherein the wavefront sensor includes a Shack-Hartmann sensor.

23. A method comprising:
(a) focusing first excitation light with a first objective lens to a first focus within a sample;
(b) scanning the first focus within a volume in the sample with one or more first scanning optical elements;
(c) de-scanning, with the one or more first scanning optical elements, as the first focus is scanned within the volume, first signal li

Assignees

• Hughes Howard Med Inst

Inventors

• Betzig Robert E
• Wang Kai

Classifications

• G01N2021/6419

  Excitation at two or more wavelengths · CPC title

• G01N2201/0675

  SLM · CPC title

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• G02B21/361

  Optical details, e.g. image relay to the camera or image sensor (G02B21/364 takes precedence; illumination details G02B21/06 and subgroups) · CPC title

• G02B21/008

  Details of detection or image processing, including general computer control · CPC title