Multifocal method and apparatus for stabilization of optical systems

Patent metadata

Field	Value
Publication number	US-10429628-B2
Application number	US-201615568770-A
Country	US
Kind code	B2
Filing date	Apr 22, 2016
Priority date	Apr 23, 2015
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

Methods and apparatus for deep microscopic super resolution imaging use two independent and variable focal planes. Movements of fiducial markers imaged using one focal plane are monitored and used to provide real-time or near real-time correction for sample drift. A second focal plane may be used to collect light for super-resolution imaging of a sample. A prototype embodiment has produced low drift when imaging many microns deeper than the fiducial markers.

First claim

What is claimed is:

1. A method for imaging a sample, the method comprising:
providing one or more fiducial markers near the sample;
imaging the sample using a first imaging system comprising an objective lens; and
while imaging the sample:
imaging the one or more fiducial markers with a second imaging system by way of the objective lens and focusing the second imaging system independently of the first imaging system;
processing images of the one or more fiducial markers obtained by the second imaging system to yield a measure of drift of the fiducial markers relative to the objective lens; and
controlling an actuator to correct for the drift.

2. A method according to claim 1 wherein imaging the sample comprises illuminating the sample with light of a first wavelength and detecting light of one or more second wavelengths different from the first wavelength.

3. A method according to claim 2 , comprising directing the light of the first wavelength for oblique incidence on the sample.

4. A method according to claim 2 , wherein imaging the fiducial markers comprises illuminating the fiducial markers with light of a fourth wavelength different from the first and second wavelengths and detecting light of a third wavelength.

5. A method according to claim 4 , wherein illuminating the one or more fiducial markers with light of the fourth wavelength comprises passing light from a second laser source through the objective lens and wherein illuminating the sample with light of the first wavelength comprises passing light from a first laser source through the objective lens.

6. A method according to claim 1 , comprising providing an asymmetrical optical element in an imaging path of the second imaging system wherein processing images of the one or more fiducial markers comprises determining a distortion in the images of the fiducial markers due to astigmatism and determining a component of the drift in a direction along a z-axis parallel to an optical axis of the objective lens based on the distortion.

7. A method according to claim 6 , wherein determining the distortion comprises determining an aspect ratio of height to width in images of the fiducial markers and if the aspect ratio is less than a reference value operating the actuator to move the sample in one direction along the z-axis and if the aspect ratio is greater than the reference amount operating the actuator to move the sample in a second direction opposite to the first direction along the z-axis.

8. A method according to claim 1 , wherein the actuator comprises a stage operative to independently control a position of the sample relative to the objective lens in two dimensions orthogonal to an optical axis of the objective lens.

9. A method according to claim 1 , wherein the second imaging system is selective for light having first wavelength characteristics and the first imaging system is insensitive to light having the first wavelength characteristics.

10. A method according to claim 1 , wherein imaging the sample comprises focusing the first imaging system on a sample plane of the sample, and imaging the fiducial markers comprises focusing the second imaging system on a fiducial marker plane of the one or more fiducial markers wherein the sample plane is spaced apart from the marker plane by at least 500 nm.

11. A method according to claim 10 wherein the sample plane is deeper than the fiducial marker plane.

12. A method according to claim 10 , wherein the fiducial marker plane coincides with a surface of a coverslip.

13. A method according to claim 1 , comprising automatically focusing the second imaging system on the one or more fiducial markers.

14. A method according to claim 1 , wherein the one or more fiducial markers comprise a plurality of fiducial markers and processing images of the one or more fiducial markers obtained by the second imaging system to yield a measure of drift comprises separately determining a drift of each of the plurality of fiducial markers and averaging the drifts.

15. A method according to claim 1 , wherein imaging the sample comprises collecting light over a period of at least 2 minutes and imaging the fiducial markers using an exposure time of less than 500 ms.

16. A method according to claim 1 , comprising providing an electrically tunable lens between the objective lens and the second imaging system and, while imaging the sample with the first imaging system, adjusting the tunable lens to focus the second imaging system on the fiducial markers.

17. A method according to claim 1 , comprising while imaging the sample, gradually increasing an exposure time of the second imaging system.

18. A method according to claim 1 , comprising while imaging the sample, gradually increasing a gain of the second imaging system.

19. A method according to claim 1 , wherein imaging the fiducial markers comprises illuminating the fiducial markers with light from a light source, and while imaging the sample, increasing an intensity of the light source.

20. A method according to claim 1 , wherein the first and second imaging systems are the same system, and imaging the sample comprises alternating the focus of the imaging system between the fiducial markers and the sample.

21. A method for stabilizing an image generated by an optical microscope, the method comprising:
illuminating a sample and a fiducial element held on a nanopositioning stage, the sample comprising a target element having a positional drift;
detecting photons from the fiducial element and the target element by independently focusing the photons from the fiducial element and the target element onto independent first and second image sensors, wherein the photons emitted by the target element and the fiducial element are respectively detected by the independent first and second image sensors allowing stabilization when the target element and the fiducial element are located at a different depths;
providing an adjustable relay lens in an optical path of one of the first and second image sensors and controlling the relay lens to focus the first and second image sensors at different focal planes;
correcting the positional drift of the sample by processing an output of the second image sensor using an algorithm configured to calculate changes in location of the fiducial element; and
performing closed-loop feedback control of the nanopositioning stage using the calculated changes in location of the fiducial element, thereby stabilizing an image of the sample.

22. A method according to claim 21 , wherein the image of the sample is a two-dimensional or three-dimensional image and the method comprises stabilizing the image in three dimensions.

23. A method according to claim 21 , wherein the nanopositioning stage is a three-axis nanopositioning stage.

24. A method according to claim 21 , wherein the fiducial element is affixed to a coverslip and the sample is imaged through the coverslip.

25. A method according to claim 21 , wherein the target element and the fiducial element are spaced apart along a z direction aligned with an optical axis of the optical microscope.

26. A method of stabilizing an image generated by an optical microscope comprising:
applying a light source to a sample and a fiducial element held on a nanopositioning stage, the sample comprising a target element and having a positional drift;
detecting photons emitted from the fiducial element with a fi

Assignees

• Univ British Columbia

Inventors

• Chou Keng C
• Tafteh Reza

Classifications

• G02B21/16

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

• G06T2207/10064

  Fluorescence image · CPC title

• G02B27/32 Primary

  Fiducial marks and measuring scales within the optical system · CPC title

• G01N2021/6463

  Optics · CPC title

• G02B27/0075

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