Method and apparatus for tracking a particle, particularly a single molecule, in a sample

Patent metadata

Field	Value
Publication number	US-9291562-B2
Application number	US-201314050583-A
Country	US
Kind code	B2
Filing date	Oct 10, 2013
Priority date	Nov 15, 2011
Publication date	Mar 22, 2016
Grant date	Mar 22, 2016

Abstract

For the purpose of tracking a movement of a particle in a sample, the particle is driven by light to emit photons, and the photons emitted by the particle are detected. The light applied to the sample features a light intensity distribution with a spatially limited minimum. The particle is tracked with the minimum of the light intensity distribution by moving the light intensity distribution with respect to the sample such that a rate of photons emitted by the particle remains minimal, and by taking an actual position of the minimum of the light intensity distribution in the sample as an actual position of the particle in the sample.

First claim

The invention claimed is:

1. A method of tracking a movement of a particle in a sample, the method comprising:
providing light;
selecting the particle from a group of particles which are driven to emit photons when subjected to the light;
forming the light to provide a light intensity distribution comprising a spatially limited minimum;
applying the light intensity distribution to the sample such that the particle is located in the spatially limited minimum of the light intensity distribution;
detecting the photons emitted by the particle;
tracking the movement of the particle with the spatially limited minimum of the light intensity distribution by
moving the light intensity distribution with respect to the sample such that a rate of the photons emitted by the particle remains minimal, and
taking an actual position of the spatially limited minimum of the light intensity distribution in the sample as an actual position of the particle in the sample.

2. The method of claim 1 , wherein the spatially limited minimum of the light intensity distribution is spatially limited in at least one spatial dimension; and wherein the particle is tracked with the spatially limited minimum of the light intensity distribution in all directions of all dimensions in which the spatially limited minimum is spatially limited.

3. The method of claim 1 , wherein the light is formed to provide the light intensity distribution in that wavefronts of a coherent beam of the light are modulated, and in that the beam of the light with the modulated wavefronts is focussed into the sample to provide the spatially limited minimum of the light intensity distribution as a point of essentially zero intensity of an interference pattern.

4. The method of claim 3 , wherein a modulation of the wavefronts is dynamically varied such that the spatially limited minimum of the light intensity distribution is alternately spatially limited in different spatial dimensions.

5. The method of claim 1 , wherein the light is formed to provide the light intensity distribution in that at least two coherent beams of the light are focussed into a same focal region in the sample to provide the spatially limited minimum of the light intensity distribution as a point of essentially zero intensity of an interference pattern.

6. The method of claim 1 , wherein the light is formed to provide the light intensity distribution such that a spatial position of the spatially limited minimum does not vary with a wavelength of the light.

7. The method of claim 1 , wherein the photons are detected by a point detector.

8. The method of claim 1 , wherein the photons are detected by at least two adjacent point detectors.

9. The method of claims 8 , wherein a direction of an initial movement of the particle is determined from relative numbers of photons emitted by the particle and detected by the adjacent point detectors.

10. The method of claim 1 , wherein the sample is imaged with a camera.

11. The method of claim 10 , wherein a starting position of the particle is determined with the camera when the sample is uniformly subjected to the light.

12. The method of claim 10 , wherein the actual position of the spatially limited minimum of the light intensity distribution in the sample is determined on a basis of positions at which the light reflected by the sample is detected with the camera.

13. The method of claim 10 , wherein a direction of an initial movement of the particle is determined on a basis of positions at which the photons emitted by the particle are detected by the camera.

14. The method of claim 1 , wherein an actual position of the minimum spatially limited of the light intensity distribution in the sample is determined on a basis of actual settings of scanners used for moving the light intensity distribution with respect to the sample.

15. The method of claim 1 , wherein the light is applied to the sample in pulses and wherein the photons emitted by the particle are detected in a limited time interval after each of the pulses.

16. The method of claim 15 , wherein the light is selected from white light.

17. The method of claim 1 , wherein the particle is selected from a group of particles which are driven to emit photons when subjected to the light by a multiphoton process.

18. The method of claim 1 , wherein the photons emitted by the particle are analysed for determining at least one feature selected from a group of features consisting of
wavelength,
polarisation,
absolute rate,
relative numbers detected by adjacent point detectors,
coincidence, and
detection time point after each pulse of the light.

19. The method of claim 1 , wherein photons emitted out of a detection volume including the spatially limited minimum of the light intensity distribution are analysed for determining at least one feature selected from a group of features consisting of
wavelength,
polarisation,
absolute rate,
relative numbers detected by adjacent point detectors,
coincidence, and
detection time point after each pulse of the light, and

wherein the determined at least one feature is checked for compliance with a single particle emitting the analysed photons.

20. The method of claim 1 , wherein a number of particles in the sample, which belong to the group of particles, is reduced by bleaching at least one particle of the number of particles.

21. The method of claim 1 , wherein the particle is selected from a subgroup of particles which are activatable from a first state in which the particles of the subgroup can not be driven to emit photons by the light into a second state in which the particles of the subgroup can be driven to emit photons by the light.

22. The method of claim 21 , wherein the particle is selected from a sub-subgroup of particles which are activatable from the first state into the second state by activation light in a multiphoton process, and wherein the particle is activated by activation light focussed into the sample.

23. The method of claim 1 , wherein a switch-off signal is provided with a signal intensity distribution enclosing the light intensity distribution, the switch-off signal switching-off other particles belonging to the group of particles which are driven to emit photons when subjected to the light.

24. The method of claim 1 , wherein the photons emitted by the particle and detected are counted and wherein an absolute number of the photons, that have already been emitted by the particle and detected, is indicated.

25. The method of claim 1 , wherein at least one further particle is selected from the group of particles which are driven to emit photons when subjected to the light; and wherein movements of the particle and of the at least one further particle are alternately tracked with the spatially limited minimum of the light intensity distribution.

26. The method of claim 1 , wherein further light differing from the light in at least one light feature selected from wavelength and polarization of at least one light component is provided, wherein a further particle is selected from a further group of particles which are driven to emit photons when subjected to the further light; wherein the further light is formed to provide a further light intensity distribution comprising a further spatially limited minimum; wherein the further light intensity distribution is applied to the sample such that the further particle is l

Assignees

• Max Planck Gesellschaft

Inventors

• Hell Stefan W

Classifications

• G01N2015/0046

  in gas, e.g. smoke · CPC title

• G01N15/1434

  Optical arrangements · CPC title

• G01N21/6458 Primary

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

• G01N21/6486

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• G01N21/64 Primary

  Fluorescence; Phosphorescence · CPC title