Laser scanning microscope and method for determining a position of a fluorophore

Patent metadata

Field	Value
Publication number	US-12111454-B2
Application number	US-202017779592-A
Country	US
Kind code	B2
Filing date	Nov 18, 2020
Priority date	Nov 29, 2019
Publication date	Oct 8, 2024
Grant date	Oct 8, 2024

Abstract

A laser scanning microscope includes a light source configured to emit an illumination light beam. The illumination light beam has a transverse light intensity profile comprising an intensity minimum. The laser scanning microscope further includes a scanning device configured to scan the illumination light beam along a closed trajectory in a target area of a specimen, and a detector configured to detect fluorescence light emitted by a fluorophore within the target area of the specimen. The fluorophore is excited by the illumination light beam. The laser scanning microscope further includes a processor configured to determine an intensity distribution of the fluorescence light as a function of time and to determine a position of the fluorophore within the target area based on the intensity distribution of the fluorescence light.

First claim

The invention claimed is:

1. A laser scanning microscope, comprising:
a light source configured to form an illumination light beam, the illumination light beam having a transverse light intensity profile comprising an intensity minimum located at a center of the illumination light beam,
a scanning device configured to scan the illumination light beam so that the center of the illumination light beam moves along a closed trajectory in a target area of a specimen,
a detector configured to detect fluorescence light emitted by a fluorophore within the target area of the specimen, the fluorophore being excited by the illumination light beam, and
a processor configured to;
determine an intensity distribution of the fluorescence light as a function of time,
fit a periodic function to the intensity distribution of the fluorescence light, and
determine a position of the fluorophore within the target area based on at least one parameter of the fitted periodic function.

2. The laser scanning microscope according to claim 1 , wherein the processor is configured to determine an amplitude spectrum, a phase spectrum and/or a frequency spectrum from the intensity distribution of the fluorescence light, to determine a number of fluorophores within the target area from the amplitude spectrum, the phase spectrum and/or the frequency spectrum, and to determine the position of each of the number of fluorophores based on the amplitude spectrum, the phase spectrum and/or the frequency spectrum.

3. The laser scanning microscope according to claim 1 , wherein the scanning device is configured such that a time interval required for the illumination light beam to run along the closed trajectory once is shorter than an average burst time of the fluorophore.

4. The laser scanning microscope according to claim 1 , wherein the closed trajectory comprises an elliptical, a circular or a spiral trajectory.

5. The laser scanning microscope according to claim 4 , wherein a dimension of the closed trajectory is smaller than or equal to a beam diameter of the illumination light beam.

6. The laser scanning microscope according to claim 1 , wherein the closed trajectory is scanned relative to a predetermined position of the target area and/or wherein a periodic recurring position of the closed trajectory is at a position of the target area or deviates from the position of the target area maximal by a beam diameter of the illumination light beam.

7. The laser scanning microscope according to claim 1 , wherein the light source comprises a phase mask for creating an optical vortex along a propagation direction of the illumination light beam.

8. The laser scanning microscope according to claim 1 , further comprising a beam splitter configured to separate the illumination light beam and the fluorescence light emitted by the fluorophore.

9. The laser scanning microscope according to claim 1 , further comprising a second scanning device for directing the illumination light beam onto the target area.

10. The laser scanning microscope according to claim 9 , wherein at least one of the second scanning device and the scanning device is operated either time sequentially or at the same time.

11. The laser scanning microscope according to claim 1 , wherein the scanning device comprises at least one of: a wobbling device, a MEMS device, an acousto-optic deflector (AOD), an acousto-optic modulator (AOM), a movable lens, or a piezo tube device.

12. The laser scanning microscope according to claim 1 , wherein the scanning device is arranged in a pupil plane of the laser scanning microscope or in a plane that is optically equivalent to the pupil plane of the laser scanning microscope.

13. The laser scanning microscope according to claim 1 , wherein the microscope is a confocal microscope.

14. The laser scanning microscope according to claim 1 , wherein the scanning device comprises a resonant MEMS mirror or a piezo driven MEMS.

15. A method for determining a position of a fluorophore within a target area of a specimen, the method comprising:
forming an illumination light beam, the illumination light beam having a transverse light intensity profile comprising an intensity minimum located at a center of the illumination light beam,
scanning the illumination light beam so that the center of the illumination light beam moves along a closed trajectory in the target area of the specimen,
exciting a fluorophore within the target area with the illumination light beam,
detecting fluorescence light emitted by the fluorophore,
determining an intensity distribution of the fluorescence light as a function of time,
fitting a periodic function to the intensity distribution of the fluorescence light, and
determining a position of the fluorophore within the target area based on at least one parameter of the fitted periodic function.

Assignees

• Leica Microsystems

Inventors

• Giske Arnold
• Foelling Jonas

Classifications

• G02B21/0072

  details concerning resolution or correction, including general design of CSOM objectives · CPC title

• G02B21/0048

  scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors · 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

• G01N21/6458

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

• G02B26/0875

  by means of one or more refracting elements · CPC title