Method of high spatial resolution determining a position of a singularized molecule which is excitable for emission of luminescence light

Patent metadata

Field	Value
Publication number	US-10900901-B2
Application number	US-201916377603-A
Country	US
Kind code	B2
Filing date	Apr 8, 2019
Priority date	Oct 10, 2016
Publication date	Jan 26, 2021
Grant date	Jan 26, 2021

Abstract

For spatial high resolution determining a position of a singularized molecule, which is excitable with excitation light for emission of luminescence light, in n spatial dimensions in a sample, a preliminary local area including the singularized molecule is determined The excitation light is directed onto the sample with an intensity distribution, which has a zero point and intensity increasing regions adjoining the zero point on both sides in each of the n spatial dimensions. At first, the zero point is arranged at preliminary positions on known sides of the preliminary local area. Then, present positions of the zero point are successively shifted into the preliminary local area in each of the n spatial dimensions depending on photons of the luminescence light which is quasi-simultaneously separately registered for the present positions of the zero point in that the zero point is repeatedly shifted between the present positions of the zero point.

First claim

We claim:

1. A method of spatial high resolution determining, in n spatial dimensions, a position of a singularized molecule in a sample, the singularized molecule being excitable with excitation light for emission of luminescence light, and n being 1, 2 or 3, the method comprising
providing the excitation light with an intensity distribution which comprises a zero point and intensity increasing regions delimiting the zero point on both sides in each of the n spatial dimensions,
determining a preliminary local area in the sample which includes the singularized molecule,
defining at least one preliminary position of the zero point per each of the n spatial dimensions which is on a known side of the preliminary local area in the respective one of the n spatial dimensions,
directing the excitation light with the intensity distribution comprising the zero point onto the sample, and
separately registering the luminescence light emitted by the singularized molecule for each of the positions of the zero point in the sample,
wherein, starting from the preliminary positions, present positions of the zero point are successively shifted into the preliminary local area in each of the n spatial dimensions depending on the photons of the luminescence light separately registered for each of the present positions of the zero point, and
wherein the luminescence light is quasi-simultaneously separately registered for the present positions of the zero point in that the zero point is repeatedly shifted between the present positions of the zero point.

2. The method of claim 1 , wherein the present positions of the zero point are successively shifted into the preliminary local area depending on
on rates or
intervals in time

at which the photons of the luminescence light are separately registered for each of the present positions of the zero point.

3. The method of claim 1 , wherein a number of the preliminary positions of the zero point is between n and 2 n.

4. The method of claim 1 , wherein, in at least one of the spatial dimensions, the position of the singularized molecule in the sample
is set to be equal to that position of the zero point associated with the respective spatial dimension for that the photons of the luminescence light have been separately registered at a smallest rate or at longest intervals in time of all positions of the zero point associated with the respective spatial dimension, or
deduced from a rate or intervals in time at which the photons of the luminescence light are separately registered for at least one final position of the zero point associated with the respective spatial dimension.

5. The method of claim 1 ,
wherein the preliminary positions include two positions of the zero point per each of the n spatial dimensions which are arranged on both sides of the preliminary local area in the respective one of the n spatial dimensions, and
wherein by the step of successively shifting the present positions of the zero point associated with all spatial dimensions depending on the photons of the luminescence light separately registered for each of the present positions of the zero point a remaining local area remaining between the present positions of the zero point and including the singularized molecule is successively reduced in size.

6. The method of claim 5 , wherein the remaining local area remaining between the present positions of the zero point and including the singularized molecule is successively reduced in size until its extensions in each of the n spatial dimensions are not longer than a predetermined precision.

7. The method of claim 6 , wherein the predetermined precision is in one of
a range from 0.5 nm to 20 nm and
a range from 1 nm to 10 nm.

8. The method of claim 1 , wherein the intensity increasing regions delimiting the zero point on both sides in each of the n spatial dimensions are symmetric with regard to the zero point.

9. The method of claim 1 , wherein at least one of the present positions of the zero point is shifted as soon as an average value of p photons of the luminescence light have been registered for all present positions, wherein p is one of
not higher than 30,
not higher than 20,
not higher than 10 and
not higher than 5.

10. The method of claim 1 , wherein at least one of the present positions of the zero point is shifted as soon as a total of n×q photons of the luminescence light have been registered for all present positions, wherein q is one of
not higher than 50,
not higher than 25 and
not higher than 5.

11. The method of claim 1 , wherein each of the present positions of the zero point is shifted as soon as m photons of the luminescence light have been separately registered for the respective one of the present positions, wherein m is one of
not higher than 30,
not higher than 20,
not higher than 10,
not higher than 5 and
not higher than 3.

12. The method of claim 1 , wherein a maximum intensity of the excitation light is set such that the preliminary local area, with regard to each of the preliminary positions of the zero point, is in a region of the intensity distribution of the excitation light of not more than 90% of a saturation intensity of the excitation light.

13. The method of claim 12 , wherein the maximum intensity of the excitation light is successively increased with shifting the present positions of the zero point.

14. The method of claim 13 , wherein the maximum intensity of the excitation light is increased such that a rate or an average interval in time at which the photons of the luminescence light are registered for all present positions of the zero point is at least temporarily kept constant.

15. The method of claim 13 , wherein the maximum intensity of the excitation light is increased by at least 50% in total.

16. The method of claim 1 , wherein, at a beginning of determining the position of the singularized molecule, a larger area of the sample including the singularized molecule is scanned with the zero point or with a Gaussian intensity distribution of the excitation light in each of the n spatial dimensions, wherein the preliminary local area is determined from a course of an intensity of the luminescence light registered during scanning.

17. The method of claim 1 , wherein, at a beginning of determining the position of the singularized molecule, the excitation light is directed with a Gaussian intensity distribution point by point or on a circular or spiral track onto a larger area of the sample including the singularized molecule, wherein the preliminary local area is determined from a course of an intensity of the luminescence light registered over the points or the spiral track, respectively.

18. The method of claim 1 , wherein, at a beginning of determining the position of the singularized molecule, a larger area of the sample including the singularized molecule is as a whole subjected to the excitation light and imaged on a spatially resolving detector, wherein the preliminary local area is determined from a spatial distribution of the luminescence light registered with the detector.

19. The method of claim 1 , wherein the luminescence light is registered at a spatial resolution, and wherein the position of the singularized molecule is additionally determined from a spatial distribution of all luminescence light which has been emitted by the singularized molecule and registered.

20. The method of claim 1 , wherein the sample, prior to determining the position of the singularized molecule, is s

Assignees

• Max Planck Gesellschaft

Inventors

• Balzarotti Francisco
• Eilers Yvan
• Gwosch Klaus
• Hell Stefan W

Classifications

• G02B21/0076

  arrangements using fluorescence or luminescence · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N21/6458 Primary

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

• G02B21/365

  Control or image processing arrangements for digital or video microscopes (G02B21/361, G02B21/362 take precedence) · CPC title