Microscopy system with auto-focus adjustment by low-coherence interferometry

Patent metadata

Field	Value
Publication number	US-9869852-B2
Application number	US-201614994835-A
Country	US
Kind code	B2
Filing date	Jan 13, 2016
Priority date	Jan 26, 2015
Publication date	Jan 16, 2018
Grant date	Jan 16, 2018

Abstract

Disclosed are several technical approaches of using low coherence interferometry techniques to create an autofocus apparatus for optical microscopy. These approaches allow automatic focusing on thin structures that are positioned closely to reflective surfaces and behind refractive material like a cover slip, and automated adjustment of focus position into the sample region without disturbance from reflection off adjacent surfaces. The measurement offset induced by refraction of material that covers the sample is compensated for. Proposed are techniques of an instrument that allows the automatic interchange of imaging objectives in a low coherence interferometry autofocus system, which is of major interest in combination with TDI (time delay integration) imaging, confocal and two-photon fluorescence microscopy.

First claim

What is claimed is:

1. A microscopy system comprising:
a low coherence interferometer;
a beam splitter that transmits light to a microscope and reflects light from the low coherence interferometer into an optical path parallel to an optical axis of an imaging objective;
wherein the imaging objective that focuses light from the low coherence interferometer into a sample, the sample being covered by a partially reflecting medium;
wherein the low coherence interferometer is configured to have a detection aperture that is not coincident with an aperture that is defined by an area with a point symmetry of an illumination aperture, the point symmetry being an intercept of the optical axis of the imaging objective with an aperture plane; and
wherein the interferometer is configured to detect a portion of back-scattered light from the sample and exclude a substantial portion of light reflected from the partially reflecting medium.

2. The system of claim 1 , wherein the illumination aperture is at least half FWHM (full width at half maximum) offset to a center of the optical axis and the detection aperture coincides with the illumination aperture.

3. The system of claim 1 , wherein the illumination and detection apertures are coincident and are clipped to avoid point symmetry with the optical axis.

4. The system of claim 1 , wherein the detection aperture is ring-shaped and is greater than the illumination aperture.

5. The system of claim 1 , wherein the illumination aperture is ring-shaped and is greater than the detection aperture.

6. The system of claim 1 , wherein the detection aperture is at an arbitrary position not coinciding with a point-symmetrical mirror image of the illumination aperture.

7. The system of claim 1 , wherein low coherence interferometer comprises a light source that is fiber coupled with an optical coupler, and light from the light source is forwarded in a fiber to a collimation objective, so that the light enters an interferometer module comprising
a beam splitting device; and
a retro-reflecting device;
wherein a portion of the light is reflected by the beam splitting device to the retro-reflecting device, back-reflected by the retro-reflecting device to the beam splitting device and then reflected by the by beam splitting device to fiber optic coupler, a light path from the beam splitting device to the retro-reflecting device forming a reference arm;
wherein another portion of the light enters an imaging path over a partially reflective mirror through the imaging objective, passes through the partially reflective surface in the sample;
wherein back-scattered light from the sample, a portion of light back-reflected by the partially reflective surface and a portion of a portion of the light back-reflected in the reference arm propagate into a spectrometer comprising a collimator, a grating, a focus optic and a linear sensor;
wherein a spectrum detected by the sensor is processed in a processing unit which determines a relative optical path length from the beam splitting device to the retro-reflecting device with an optical path length from the beam splitting device to the sample by analysis of the special frequency on the sensor, and the processing unit determines an offset of the relative optical path length based on a calibrated focus set path length and drives a motor unit to move a sample holder so that the sample on the sample holder is brought into the focus of the imaging objective.

8. The system of claim 7 , wherein the imaging objective is interchangeable, and the retro-reflecting device is motorized, so that the reference path length can be adjusted to compensate for optical path length differences in different objectives.

9. The system of claim 7 , wherein the imaging objective is interchangeable, and the system further comprises an interchangeable dispersion compensating glass plate that matches optical path length differences in different objectives.

10. The system of claim 7 , wherein the imaging objective is interchangeable, and the interferometer module comprises prism elements that are adjusted along their symmetry axis to match optical path length differences in different objectives.

11. The system of claim 7 , wherein the imaging objective is interchangeable, and the system is configured to compensate path length differences in different objectives numerically.

12. The system of claim 11 , wherein compensation data for path length differences in different objectives are stored in the processing unit.

13. A method for automatic relative focus adjustment of an autofocus microscopy system that comprises a low coherence interferometer and a beam splitter that transmits light to a microscope and reflects light from the low coherence interferometer into an optical path parallel to an optical axis of an imaging objective, wherein a sample is covered by a cover material, the method comprising:
computing A-scan data of a relative air/cover material position;
computing A-scan data of a relative sample position;
calibrating a focus position;
computing a thickness of the cover material;
processing a motor set position; and
commanding a movement of a motor unit;
wherein if a signal back scattered from the sample is less than 10 dB above a noise floor, storing a last position corresponding to a signal back scattered from the sample that is greater than 10 dB;
wherein if a signal back scattered from the sample is less than 10 dB above the noise floor, the last position is used for the processing.

14. A method for operating a microscopy system that comprises a low coherence interferometer and a beam splitter that transmits light to a microscope and reflects light from the low coherence interferometer into an optical path parallel to an optical axis of an imaging objective, the method comprising:
focusing light from the low coherence interferometer into a sample by the
imaging objective, the sample being covered by a partially reflecting medium;
providing to the low coherence interferometer a detection aperture that is not coincident with an aperture that is defined by an area with a point symmetry of an illumination aperture, the point symmetry being an intercept of the optical axis of the imaging objective with an aperture plane; and
detecting a portion of back-scattered light from the sample and excluding a substantial portion of light reflected from the partially reflecting medium.

15. The method of claim 14 , wherein the illumination aperture is at least half FWHM (full width at half maximum) offset to the center of the optical axis and the detection aperture coincides with the illumination aperture.

16. The method of claim 14 , wherein the illumination and detection apertures are coincident and are clipped to avoid point symmetry with the optical axis.

17. The method of claim 14 , wherein the detection aperture is ring-shaped and is greater than the illumination aperture.

18. The method of claim 14 , wherein the illumination aperture is ring-shaped and is greater than the detection aperture.

19. The method of claim 14 , wherein the detection aperture is at an arbitrary position not coinciding with a point-symmetrical mirror image of the illumination aperture.

Assignees

• Thorlabs Inc

Inventors

• Cable Alex
• Wollenzin Jörn
• Johnstone Ross
• Gossage Kirk
• Brooker Jeffrey S
• Mills Jason
• Jiang James
• Hillmann Dierck

Classifications

• G01B9/02077

  of the object · CPC title

• G01B9/02091

  Tomographic interferometers, e.g. based on optical coherence · CPC title

• G02B21/0004

  specially adapted for specific applications · CPC title

• G01B9/02063

  by particular alignment of focus position, e.g. dynamic focussing in optical coherence tomography · CPC title

• G01B2290/50

  Pupil plane manipulation, e.g. filtering light of certain reflection angles · CPC title