Resolution enhancement for light sheet microscopy systems and methods

Patent metadata

Field	Value
Publication number	US-10401604-B2
Application number	US-201515511580-A
Country	US
Kind code	B2
Filing date	Sep 24, 2015
Priority date	Sep 24, 2014
Publication date	Sep 3, 2019
Grant date	Sep 3, 2019

Abstract

Embodiments of a resolution enhancement technique for a light sheet microscopy system having a three objective lens arrangement in which one objective lens illuminates a sample and the second and third objective lenses collect the fluorescence emissions emitted by the sample are disclosed. The second objective lens focuses a first portion of the fluorescence emissions for detection by a second detection component, while the third objective lens focuses a second portion of the fluorescence emissions through a diffractive or refractive optic component for detection by a first detector component. A processor combines the images resulting from the first and second portions of the fluorescence emissions for generating composite images with increased axial and lateral resolution.

First claim

What is claimed is:

1. A light sheet microscopy system comprising:
a light source for transmitting a single light beam;
a first optics arrangement for relaying the single light beam and transforming the single light beam into a light sheet;
a first objective lens for focusing the light sheet into a sample for generating a first portion of fluorescence emissions and a second portion of the fluorescence emissions, the sample defining a plane;
a second objective lens in perpendicular orientation relative to the first objective lens, the second objective lens collecting a first portion of the fluorescence emissions;
a first detection component for receiving the first portion of the fluorescence emissions received from the second objective lens;
a third objective lens in perpendicular relation to the plane of the sample, the third objective lens being aligned to receive the second portion of the fluorescence emissions;
an optical system in operative communication with the third objective lens for extending the axial resolution and preserving the lateral resolution of the second portion of the fluorescence emissions;
a second detection component for receiving the second portion of the fluorescence emissions simultaneously as the first portion of the fluorescence emissions is received by the first detector; and
a processor in operative communication with the first detection component and the second detection component for combining the first portion of the fluorescence emissions with the second portion of the fluorescence emissions to generate a composite image having an extended axial and lateral resolution.

2. The microscopy system of claim 1 , wherein the optical system further comprises a diffractive optic that introduces aberrations to the second portion of the fluorescence emissions.

3. The microscopy system of claim 1 , wherein the optical system further comprises a refractive optic having a plurality of incoherent apertures.

4. The microscopy system of claim 1 , wherein the optical system comprises an extended depth-of-focus optic arrangement that extends either the axial resolution or the lateral resolution of the second portion of the fluorescence emissions.

5. The microscopy system of claim 4 , wherein the third objective lens is oscillated to extend either the axial resolution or the lateral resolution of the second portion of the fluorescence emissions.

6. The microscopy system of claim 1 , wherein the first detection component comprises a first camera and the second detection component comprises a second camera, wherein the microscopy system further comprising a second optic arrangement for focusing the second portion of fluorescence emissions onto the second detection component and wherein the second optic arrangement comprises a first lens and a second lens in 4f telescopic relation to image the back focal plane of the third objective lens at the back focal plane of the second lens.

7. The microscopy system of claim 1 , wherein the optical system comprises a cubic phase plate component.

8. The microscopy system of claim 1 , wherein the optical system comprises an incoherent layer cake component.

9. The microscopy system of claim 1 , wherein the optical system comprises a multi-focus grating component.

10. A method comprising:
transmitting a single light beam from an illumination source;
transforming the single light beam into a light sheet;
focusing the light sheet into a sample for generating fluorescence emissions, the sample defining a plane;
positioning a first objective lens in perpendicular relation to a second objective lens, wherein the first objective lens illuminates the sample with the light sheet for generating a first portion of fluorescence emissions and a second portion of fluorescence emissions and the second objective lens collects the first portion of the fluorescence emissions;
detecting the first portion of the fluorescence emissions received from the second objective lens;
positioning a third objective lens in perpendicular relation to the plane of the sample, the third objective lens being positioned to receive a second portion of the fluorescence emissions;
extending the axial resolution of the second portion of the fluorescence emissions and preserving the lateral resolution of the second portion of the fluorescence emissions;
detecting the second portion of the fluorescence emissions simultaneously as the first portion of the fluorescence emissions is detected by the first detector; and
combining the first portion of the fluorescence emissions with the second portion of the fluorescence emissions to generate a composite image having an extended axial and lateral resolution.

11. The method of claim 10 , wherein extending the axial resolution while preserving the lateral resolution of the second portion of the fluorescence emissions comprises positioning a diffractive optic in relation to the third objective lens.

12. The method of claim 10 , wherein extending the axial resolution while preserving the lateral resolution of the second portion of the fluorescence emissions comprises positioning a refractive optic in relation to the third objective lens.

13. The method of claim 10 , further comprising:
positioning a first lens in 4f telescopic relation with a second lens so as to image a back focal plane of the third objective lens at the back focal plane of the second lens.

14. The method of claim 13 , further comprising:
positioning a third lens in 4f telescopic relation with the second lens.

15. A light sheet microscopy system comprising:
a light source for transmitting a single light beam;
a first optics arrangement for relaying the single light beam and transforming the single light beam into a light sheet;
a first objective lens for focusing the light sheet into a sample for generating a first portion of fluorescence emissions and a second portion of fluorescence emissions, the sample defining a plane;
a second objective lens in perpendicular orientation relative to the first objective lens, the second objective lens collecting a first portion of the fluorescence emissions;
a first detection component for receiving the first portion of the fluorescence emissions received from the second objective lens;
a third objective lens aligned to receive a second portion of the fluorescence emissions;
a depth of focus optic component in operative communication with the third objective lens for introducing aberrations in the second portion of the fluorescence emissions;
a second detection component for receiving the second portion of the fluorescence emissions simultaneously as the first portion of the fluorescence emissions is detected by the first detector; and
a processor in operative communication with the first detection component and the second detection component for combining the first portion of the fluorescence emissions with the second portion of the fluorescence emissions to generate a composite image with increased resolution.

16. The light sheet microscopy system of claim 15 , wherein introducing aberrations in the second portion of the fluorescence emissions degrades the axial resolution of the second portion of the fluorescence emissions.

17. The light sheet microscopy system of claim 15 , wherein introducing aberrations in the second portion of the fluorescence emissions substantially preserves the lateral resolution of the second portion of the fluorescence emissions.

18. The light sheet microscopy system of claim 15 , wherein the composite image combines the axial resolution of the f

Assignees

• Us Health
• Shroff Hari
• Wu Yicong
• Abrahamsson Sara

Inventors

• Shroff Hari
• Wu Yicong
• Abrahamsson Sara

Classifications

• G02B27/42

  Diffraction optics {, i.e. systems including a diffractive element being designed for providing a diffractive effect}(G02B27/60 takes precedence) · CPC title

• G02B27/0075

  with means for altering, e.g. increasing, the depth of field or depth of focus · CPC title

• G02B21/16 Primary

  adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)} · CPC title

• G02B21/10

  affording dark-field illumination (G02B21/14 {and G02B21/125} take precedence) · CPC title

• G02B21/0076

  arrangements using fluorescence or luminescence · CPC title