Method of and apparatus for spatially measuring nano-scale structures
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.
1. A method of spatially measuring a plurality of nano-scale structures in a sample, the method comprising the steps of:
marking each of 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 in the sample are determined in n spatial directions, and 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 different positions of the minimum.
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 structures, 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. The method of claim 1 , wherein the positions of the individual fluorescent markers in the sample are determined in n spatial directions, and wherein the positions of the individual fluorescent markers are determined from the intensities of the fluorescence light which are registered for not more than 3n different positions of the minimum.
15. The method of claim 14 , 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.
16. The method of claim 15 , 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.
17. The method of claim 1 , wherein the positions of the individual fluorescent markers in the sample are determined in n spatial directions, and wherein the positions of the individual fluorescent markers are determined from the intensities of the fluorescence light which are registered for not more than 2n different positions of the minimum.
18. The method of claim 17 , 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.
19. The method of claim 18 , 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.
20. 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,
wherein the positioning aids comprise light reflecting entities selected from gold particles, silver particles and quantum dots,
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.