Method of and Apparatus for Spatially Measuring Nano-Scale Structures

Patent metadata

Field	Value
Publication number	US-2018259458-A1
Application number	US-201815912928-A
Country	US
Kind code	A1
Filing date	Mar 6, 2018
Priority date	Mar 7, 2017
Publication date	Sep 13, 2018
Grant date	—

Abstract

A method of spatially measuring a plurality of nano-scale structures in a sample comprises the steps of: marking the individual structures at different locations with fluorescent markers, coupling the individual structures to individual positioning aids whose positions in the sample are known, exciting the fluorescent markers with excitation light for emission of fluorescence light, wherein an intensity distribution of the excitation light has a local minimum, arranging the local minimum at different positions in a close-up range around the position of respective positioning aid whose dimensions are not larger than the diffraction limit at the wavelength of the excitation light, registering the fluorescence light emitted out of the sample separately for the individual fluorescent markers and for the different positions of the minimum, and determining positions of the individual fluorescent markers in the sample from the intensities of the fluorescence light registered.

First claim

We claim:

1 . A method of spatially measuring a plurality of nano-scale structures in a sample, the method comprising the steps of:
marking the individual structures at different locations with fluorescent markers, coupling the individual structures to individual positioning aids whose positions in the sample are
known or
determined after the step of coupling from light reflected by the positioning aids,
exciting the fluorescent markers with excitation light for emission of fluorescence light, wherein
either an intensity distribution of the excitation light
or an intensity distribution of further light which has an influence on the emission of fluorescence light by the fluorescent markers

comprises a local minimum which is arranged at different positions in the sample,
registering fluorescence light emitted out of the sample separately both
for the individual fluorescent markers and
for the different positions of the minimum, and

determining positions of the individual fluorescent markers in the sample from intensities of the fluorescence light registered for the respective fluorescent marker for the different positions of the minimum,
wherein the step of exciting includes arranging the local minimum at the different positions in a close-up range around the position of the respective positioning aid, dimensions of the close-up range not being larger than the diffraction limit at the wavelength of the excitation light and the wavelength of the fluorescence light.

2 . The method of claim 1 , wherein the dimensions of the close-up range are not larger than a half or a quarter of the diffraction limit at the wavelength of the excitation light and the wavelength of the fluorescence light.

3 . The method of claim 1 , wherein the positions of the individual fluorescent markers are determined from the intensities of the fluorescence light which are registered for not more than 4n or for not more than 3n or for not more than 2n different positions of the minimum, wherein n is the number of the spatial directions in which the positions of the individual fluorescent markers in the sample are determined.

4 . The method of claim 3 , wherein the step of determining the positions of the individual fluorescent markers in the sample includes fitting a spatial function comprising a local extremum to the intensities of the fluorescence light registered for the respective fluorescent marker for the different positions of the minimum.

5 . The method of claim 4 , wherein the spatial function comprising the local extremum is determined by scanning
at least one of the light reflecting positioning aids or at least one of the fluorescent markers or a further fluorescent marker
with the minimum and registering the light reflected out of the sample or the fluorescence light emitted out of the sample with temporal resolution while scanning.

6 . The method of claim 1 , wherein the fluorescent markers by which the individual structures are marked at different locations are selected from
fluorescent markers that differ in spectral properties selected from excitation spectra and emission spectra of the fluorescent markers, and fluorescent markers that have an active state in which they are excitable with excitation light for emission of fluorescence light and an inactive state in which they are, at least with the same excitation light, not excitable for emission of fluorescence light, and that are transferable with switching light between their active and their inactive state.

7 . The method of claim 1 , wherein the positions of the positioning aids are approached with the minimum by other means for relative movement of the minimum with regard to the sample than the means used for positioning the minimum within the close-up ranges.

8 . The method of claim 7 , wherein the positions of the positioning aids are fixed relative to fixed points of the sample and that the positions of the positioning aids are approached relative to the fixed points of the sample.

9 . The method of claim 1 , wherein the positions of the positioning aids are arranged
at minimum distances which are not smaller than twice the diffraction limit at the wavelength of the excitation light and the wavelength of the fluorescence light and in a predefined pattern in the sample selected from periodical, hexagonal and square patterns.

10 . The method of claim 1 , wherein the positioning aids in the sample are fixed coupling sites to which the structures are coupled via an immunoreaction.

11 . The method of claim 1 , wherein the positioning aids comprise light reflecting entities selected from gold particles, silver particles and quantum dots.

12 . The method of claim 1 , wherein the structures include equal structure, and wherein the equal structures are subjected to different surrounding conditions in different areas of the sample.

13 . The method of claim 1 , wherein the positions of the individual fluorescent markers in the sample are determined for at least two different points in time from the intensities of the fluorescence light registered for these at least two points in time, and wherein
the structures are subjected to different surrounding conditions at the at least two points in time or the at least two points in time succeed to an alteration of the surrounding conditions to which the structures are subjected.

14 . An apparatus for spatially measuring a plurality of nano-scale structures in a sample, wherein each of the structures is coupled to a positioning aid whose position in the sample is known or determined from light reflected by the positioning aids, the apparatus comprising
a sample holder for the sample, an objective lens directed onto the sample holder, a light source
either coupling excitation light into the objective lens in such a way that an intensity distribution of the excitation light focused by the objective lens comprises a local minimum
or coupling excitation light and further light that has an influence on the emission of fluorescence light by the fluorescent markers into the objective lens in such a way that an intensity distribution of the further light focused by the objective lens comprises a local minimum,
a scanner configured to move a position of the minimum with regard to the sample holder, a detector configured to register fluorescence light emitted out of the sample separately for the different positions of the minimum, an evaluation unit configured to determine positions of individual fluorescent markers in the sample from the intensities of the fluorescence light registered for the respective fluorescent marker for the different positions of the minimum, and a controller configured to arrange the local minimum at different positions in a close-up range around the position of the respective positioning aid, dimensions of the close-up range not being larger than the diffraction limit at the wavelength of the excitation light and the wavelength of the fluorescence light.

15 . A method of spatially measuring a nano-scale structure in a sample, the method comprising the steps of:
marking the structure at different locations with fluorescent markers, exciting the fluorescent markers with excitation light for emission of fluorescence light at the respective position of a local minimum of an intensity distribution of fluorescence inhibition light, wherein the local minimum is arranged at different positions in a close-up range in the sample whose dimensions are not larger than the diffraction limit at the wavelength of the excitation light and the wavelength of t

Assignees

• Max Planck Gesellschaft

Inventors

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

Classifications

• G02B21/0076

  arrangements using fluorescence or luminescence · CPC title

• G01N21/643

  non-biological material · CPC title

• G01N21/6456 Primary

  Spatial resolved fluorescence measurements; Imaging · CPC title

• G02B21/0036

  Scanning details, e.g. scanning stages · CPC title

• G01N21/6458 Primary

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