Multifocal macroscope for large field of view imaging of dynamic specimens

Patent metadata

Field	Value
Publication number	US-11921271-B2
Application number	US-202117326021-A
Country	US
Kind code	B2
Filing date	May 20, 2021
Priority date	May 22, 2020
Publication date	Mar 5, 2024
Grant date	Mar 5, 2024

Abstract

Provided herein is a macroscope comprising an objective apparatus comprising a multifocal widefield optics comprising a plurality of optical components configured to focus on a plurality of planes. Also provided herein are methods for analyzing a three-dimensional specimen, the method comprising obtaining, via a macroscope, synchronous multifocal optical images of a plurality of planes of the three-dimensional specimen, wherein the macroscope comprises an objective apparatus comprising a multifocal widefield optics comprising a plurality of optical components configured to focus on a plurality of planes. The three-dimensional specimen can be a biological specimen, such as brain.

First claim

We claim:

1. A macroscope comprising an objective apparatus comprising a multifocal widefield optics for a single detection path, the multifocal widefield optics comprising a multi-focus lenslet array comprising two or more lenses configured to simultaneously focus on two or more planes.

2. The macroscope of claim 1 , wherein multifocal optics comprises a dual-focus array of lenses configured to focus on two planes.

3. The macroscope of claim 1 , wherein the multifocal optics comprises an array of lenses configured to focus on more than two planes.

4. The macroscope of claim 1 , further comprising a camera configured to simultaneously capture a plurality of images captured by the macroscope, wherein the plurality of images are focused on the plurality of planes.

5. The macroscope of claim 4 , wherein the camera comprises:
a) a field of view of at least 1 cm in the longest dimension;
b) a frame rate of greater than 10 Hz; and/or
c) a pixel size of between 10 μm to 20 μm.

6. The macroscope of claim 4 operably connected to a processor and a non-transitory machine-readable medium encoding instructions, which when executed by the processor, cause the processor to process the plurality of images captured by the camera.

7. The macroscope of claim 6 , wherein the non-transitory machine-readable medium encodes instructions, which, when executed by the processor, cause the processor to merge the focused regions from the plurality of images captured by the camera to produce an image that is focused in substantially the entire field of view.

8. A method for analyzing a three-dimensional specimen, the method comprising obtaining, via a macroscope, synchronous multifocal optical images of a plurality of planes of the three-dimensional specimen, wherein the macroscope comprises an objective apparatus comprising a multifocal widefield optics for a single detection path, wherein the multifocal widefield optics comprises a multi-focus lenslet array comprising two or more lenses configured to simultaneously focus on two or more planes.

9. The method of claim 8 , wherein the multifocal optics comprises a dual-focus array of lenses configured to focus on two planes.

10. The method of claim 8 , wherein the multifocal optics comprises an array of lenses configured to focus on more than two planes.

11. The method of claim 8 , wherein the macroscope further comprises a camera configured to simultaneously capture a plurality of images captured by the macroscope, wherein the plurality of images are focused on the plurality of planes.

12. The method of claim 8 , wherein the camera comprises:
a) a field of view of at least 1 cm in the longest dimension;
b) a frame rate of greater than 10 Hz; and/or
c) a pixel size of between 10 μm to 20 μm.

13. The method of claim 11 , wherein the macroscope is operably connected to a processor and a non-transitory machine-readable medium encoding instructions, which when executed by the processor, cause the processor to process the plurality of images captured by the camera.

14. The method of claim 13 , wherein the machine-readable medium encodes instructions, which when executed by the processor, cause the processor to merge the focused regions from the plurality of images to produce an image that is focused in substantially entire field of view.

15. The method of claim 13 , wherein the machine-readable medium encodes instructions, which when executed by the processor, cause the processor to extract information of interest from the plurality of images focused on a plurality of planes without making a single-focused image.

16. The method of claim 8 , wherein the three-dimensional specimen is a biological tissue and obtaining optical images of the plurality of planes of the biological tissue indicates cellular activity in the biological tissue.

17. The method of claim 16 , wherein the biological tissue is selected from brain, placenta, eyes, pineal gland, pituitary gland, thyroid gland, parathyroid glands, thorax, heart, lung, esophagus, thymus gland, pleura, adrenal glands, appendix, gall bladder, urinary bladder, large intestine, small intestine, kidneys, liver, pancreas, spleen, stoma, ovaries, uterus, testis, skin, a cultured organoid, and a cultured cell.

18. The method of claim 17 , wherein the biological specimen is an organoid or a cultured cell, the method further comprising contacting the organoid or the culture cell with a compound and imaging the organoid or the culture cell cellular activity in the biological specimen.

19. The method of claim 8 , wherein the three-dimensional specimen comprises particles in motion and the optical imaging of the three-dimensional specimen indicates the movement of the particles in the specimen.

20. The method of claim 8 , wherein the three-dimensional specimen has a thickness of between 1 and 5 mm.

Assignees

• Univ Leland Stanford Junior
• The Board Of Trustees Of The Leland Stanford Junior Univeristy

Inventors

• Wetzstein Gordon
• Machado Tim
• Deisseroth Karl A
• Kauvar Isaac

Classifications

• G02B21/0028 Primary

  specially adapted for specific applications, e.g. for endoscopes, ophthalmoscopes, attachments to conventional microscopes · CPC title

• G02B21/006

  focusing arrangements; selection of the plane to be imaged · CPC title

• G02B21/008

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

• G02B2207/114

  Two photon or multiphoton effect · CPC title

• G02B21/365

  Control or image processing arrangements for digital or video microscopes (G02B21/361, G02B21/362 take precedence) · CPC title