Methods and systems for multidimensional imaging

Patent metadata

Field	Value
Publication number	US-11714271-B2
Application number	US-202217691731-A
Country	US
Kind code	B2
Filing date	Mar 10, 2022
Priority date	Sep 14, 2020
Publication date	Aug 1, 2023
Grant date	Aug 1, 2023

Abstract

A multi-depth confocal imaging system includes at least one light source configured to provide excitation beams and an objective lens. The excitation beams are focused into a sample at a first plurality of focus depths along an excitation direction through the objective lens. An image sensor receives emissions from the sample via the objective lens, wherein the emissions define foci relative to the image sensor at a second plurality of focus depths.

First claim

The invention claimed is:

1. A multi-depth confocal imaging system, comprising:
at least one light source configured to provide excitation beams, wherein the at least one light source is coupled to optic fibers that introduce the excitation beams into the sample, wherein the optic fibers are grouped into at least a first bundle including a first plurality of optic fibers and a second bundle including a second plurality of optic fibers, wherein the first plurality of optic fibers defines a first single focus end at a first Z axis location relative to the sample, and wherein the second plurality of optic fibers defines a second single focus end at a second Z axis location relative to the sample, and wherein the first Z axis location is offset from the second Z axis location by an offset distance;
an objective lens, wherein the excitation beams are focused into a sample at a first plurality of focus depths along an excitation direction through the objective lens, the objective lens having an optical axis; and
an image sensor that receives emissions from the sample via the objective lens, wherein the emissions define foci relative to the image sensor at a second plurality of focus depths, wherein the image sensor is oriented at an oblique orientation relative to the optical axis of the objective lens.

2. The system of claim 1 , further comprising a scanner element configured to scan the sample along a scan direction.

3. The system of claim 1 , wherein the aspect of the image sensor is positioned at an oblique orientation relative to the optical axis of the objective lens so as to compensate for the second plurality of focus depths of the emissions relative to the image sensor.

4. The system of claim 1 , wherein the light source is a laser, LED (light emitting diode), a mercury or tungsten lamp, or a super-continuous diode.

5. The system of claim 4 , wherein the laser is a continuous wave (CW) laser or a pulsed laser.

6. The system of claim 1 , wherein the light source provides excitation beams having a wavelength between 200 nm to 1500 nm.

7. The system of claim 1 , wherein the light source is a laser.

8. The system of claim 7 , wherein the laser provides excitation beams having a wavelength of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm.

9. The system of claim 1 , wherein the image sensor comprises a complementary metal-oxide-semiconductor (CMOS) array, a charge-coupled device (CCD) array, an array of photodiodes, an array of avalanche photodiodes, an array of photomultiplier tubes (PMTs), or an array of optical fibers.

10. The system of claim 1 , wherein the image sensor comprises at least one of a complementary metal-oxide-semiconductor (CMOS) array or a charge-coupled device (CCD) array.

11. The system of claim 1 , wherein the image sensor is a camera.

12. The system of claim 1 , wherein the light source is coupled through single-mode optical fibers.

13. The system of claim 1 , wherein the second plurality of focus depths correspond to the first plurality of focus depths.

14. The system of claim 1 , wherein the objective lens is an air objective lens.

15. The system of claim 1 , wherein the objective lens comprises a numerical aperture of 0.1 to 1.65.

16. The system of claim 1 , wherein the objective lens comprises a focal length of 1.6 mm to 50 mm.

17. The system of claim 1 , wherein the oblique orientation of the image sensor corresponds to an oblique orientation defined by the second plurality of focus depths of the foci.

18. The system of claim 1 , wherein the image sensor comprises an array of pixels that are oriented at the oblique orientation relative to the optical axis of the objective lens.

19. The system of claim 1 , wherein the first single focus end and the second single focus end are each at fixed locations along the Z-axis.

20. A multi-depth confocal imaging system, comprising:
at least one light source; and
optic fibers that introduce excitation beams from the at least one light source into the sample, wherein the optic fibers are grouped into at least a first bundle including a first plurality of optic fibers and a second bundle including a second plurality of optic fibers, wherein the first plurality of optic fibers defines a first single focus end at a first Z axis location relative to the sample, and wherein the second plurality of optic fibers defines a second single focus end at a second Z axis location relative to the sample, and wherein the first Z axis location is offset from the second Z axis location by an offset distance and wherein the first Z axis location and the second Z axis location collectively define an oblique tilt angle that corresponds to an oblique tilt angle by which an image sensor is oriented relative to an optical axis of an objective lens.

21. The system of claim 20 , further comprising at least one of a diffractive optical element and a micro-lens.

22. The system of claim 20 , wherein the at least one light source comprises a plurality of lasers including a first laser having a first excitation wavelength and a second laser having a second excitation wavelength,
wherein each of the lasers is coupled to a respective splitter that splits light of each laser into multiple optic fibers including a first splitter that splits light of the first laser into a first optic fiber of the first bundle and a second optic fiber of the second bundle, and a second splitter that splits light into a third optic fiber of the first bundle and a fourth optic fiber of the second bundle.

23. The system of claim 22 , wherein each bundle includes at least one optic fiber from each of the lasers.

24. The system of claim 22 , wherein the each of the first splitter and the second splitter is a 1-5 splitter, and
wherein the optic fibers are further grouped into a third bundle including a third plurality of optic fibers, a fourth bundle including a fourth plurality of optic fibers, and a fifth bundle including a fifth plurality of optic fibers;
and wherein the first splitter further splits light of the first laser into the third bundle, the fourth bundle, and the fifth bundle, and wherein the second splitter further splits light of the second laser into the third bundle, the fourth bundle, and the fifth bundle.

25. A method of generating a two- or three-dimensional image of a sample, comprising:
introducing excitation beams into a sample along an excitation direction via optic fibers, wherein the optic fibers are grouped into at least a first bundle including a first plurality of optic fibers and a second bundle including a second plurality of optic fibers, wherein the first plurality of optic fibers defines a first single focus end at a first Z axis location relative to the sample, and wherein the second plurality of optic fibers defines a second single focus end at a second Z axis location relative to the sample, and wherein the first Z axis location is offset from the second Z axis location by an offset distance;
focusing a corresponding plurality of emissions on an image sensor, wherein the emissions are focused at a plurality of depths relative to the image sensor, and wherein the image sensor is oriented at an oblique angle relative to the excitation direction; and
scanning the sample along a direction orthogonal to the excitation direction to obtain a plurality of planar images of the sample, the planar images definin

Assignees

• Singular Genomics Systems Inc

Inventors

• Cang Hu
• Glezer Eli N

Classifications

• G02B21/02 Primary

  Objectives · CPC title

• G01N21/6402

  Atomic fluorescence; Laser induced fluorescence · CPC title

• G01N21/6458 Primary

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

• G02B21/008

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