Method, computer program and apparatus for determining positions of molecules in a sample
The present disclosure relates to method, computer programs with instructions, and apparatus for determining positions of two or more spaced-apart molecules in one or more spatial directions in a sample by means of a localization microscope. The present disclosure also relates to localization microscopes using such an apparatus. Light distributions arising due to interference of coherent light are used for determining the positions of the molecules. In the method, a plurality of light distributions are generated (S 1 ) using a first light modulator having a plurality of switchable pixels. The first light modulator is arranged in an image plane of the localization microscope. Each light distribution has a local intensity minimum and regions with an intensity increase adjacent thereto. Each of the two or more molecules is illuminated (S 2 ) with one light distribution. For each of the light distributions, photons emitted by the molecules are detected (S 4 ) for different positionings of the light distribution. The light distributions are positioned (S 3 ) independently of each other. Based on the photons detected for the different positionings of the light distributions, the positions of the molecules are finally derived (S 5 ).
1. A method for determining positions of two or more spaced-apart molecules in one or more spatial directions in a sample using a localization microscope, wherein light distributions arising due to interference of coherent light are used for determining the positions of the molecules, comprising:
generating a plurality of light distributions using a first light modulator having a plurality of switchable pixels disposed in an image plane of the localization microscope, each light distribution having at least one local intensity minimum and regions with an intensity increase adjacent thereto;
illuminating each of the two or more molecules with one light distribution each;
for each of the light distributions, detecting photons emitted by the molecules for different positionings of the light distribution, the light distributions being positioned independently of each other; and
deriving the positions of the molecules based on the photons detected for the different positionings of the light distributions.
2. The method according to claim 1 , wherein grating structures or ring structures are formed by means of the switchable pixels of the first light modulator for generating the light distributions.
3. The method according to claim 2 , wherein the structures formed by means of the switchable pixels of the first light modulator are shifted for positioning the light distributions.
4. A method for determining positions of two or more spaced-apart molecules in one or more spatial directions in a sample using a localization microscope, wherein light distributions arising due to interference of coherent light are used for determining the positions of the molecules, comprising:
generating a sequence of light distributions using a first light modulator having a plurality of switchable pixels disposed in an image plane of the localization microscope, each light distribution having local intensity minima and regions with an intensity increase adjacent thereto, wherein subsequent light distributions are locally modified in accordance with estimated positions of the molecules;
illuminating the two or more molecules with the sequence of light distributions;
for each of the light distributions, detecting photons emitted by the molecules; and
deriving the positions of the molecules based on the photons detected for the different light distributions.
5. The method according to claim 4 , wherein for generating the light distributions a grating structure is formed by means of the switchable pixels of the first light modulator, and wherein subsequent light distributions are locally modified by switching off individual grating lines of the grating structure.
6. The method according to claim 5 , wherein those grating lines or groups of grating lines are switched off that cause an intensity at an estimated position of a molecule that exceeds a threshold.
7. The method according to claim 5 , wherein the grating structure formed by means of the switchable pixels of the first light modulator is shifted for subsequent light distributions.
8. The method according to claim 1 , wherein a second light modulator for influencing the light distributions is arranged in a Fourier plane of the localization microscope, which is arranged to block individual diffraction orders.
9. The method according to claim 1 , wherein the first light modulator is an amplitude modulator or a phase modulator.
10. The method according to claim 2 , wherein for successive position determinations with a light distribution, an intensity profile of the light distribution is changed by changing a structural property of the associated grating structure or ring structure.
11. The method according to claim 1 , wherein the light distributions result from a temporal superposition of different partial light distributions.
12. The method according to claim 11 , wherein the partial light distributions result from the interference of different diffraction orders generated by the first light modulator.
13. The method according to claim 1 , wherein a marker in the sample is detected using at least one light distribution for determining a drift.
14. The method according to claim 1 , wherein a drift is determined from changes in the same direction in the measured positions of molecules in successive measurements.
15. The method according to claim 1 , wherein the photons emitted by the molecules are detected using a camera or an array of photon counters.
16. An apparatus for determining positions of two or more spaced-apart molecules in one or more spatial directions in a sample using a localization microscope, wherein light distributions arising due to interference of coherent light are used for determining the positions of the molecules, comprising:
an operating unit adapted to drive a first light modulator having a plurality of switchable pixels, which is disposed in an image plane of the localization microscope, to generate a plurality of independently positionable light distributions, each light distribution having at least one local intensity minimum and regions with an intensity increase adjacent thereto; and
an evaluation unit adapted to derive the positions of the molecules on the basis of photons emitted by the molecules for each of the light distributions for different positionings of the respective light distributions.
17. An apparatus for determining positions of two or more spaced-apart molecules in one or more spatial directions in a sample using a localization microscope, wherein light distributions arising due to interference of coherent light are used for determining the positions of the molecules, comprising:
an operating unit adapted to drive a first light modulator having a plurality of switchable pixels, which is disposed in an image plane of the localization microscope, to generate a sequence of light distributions, each light distribution having local intensity minima and regions with an intensity increase adjacent thereto, wherein subsequent light distributions are locally modified in accordance with estimated positions of the molecules; and
an evaluation unit adapted to derive the positions of the molecules on the basis of photons emitted by the molecules for the different light distributions.
18. A microscope, with:
a light source for generating coherent light;
a first light modulator having a plurality of switchable pixels, which is disposed in an image plane of the microscope for generating a plurality of light distributions arising due to interference of coherent light, each light distribution having at least one local intensity minimum and regions with an intensity increase adjacent thereto;
optical means for illuminating two or more spaced-apart molecules in a sample with one light distribution each;
a detector unit for detecting photons emitted by the molecules for each of the light distributions for different positionings of the light distributions, the light distributions being positionable independently of each other; and
an apparatus according to claim 17 for determining positions of the two or more molecules in one or more spatial directions.
19. A microscope, with:
a light source for generating coherent light;
a first light modulator having a plurality of switchable pixels, which is disposed in an image plane of the microscope for generating a sequence of light distributions arising due to interference of coherent light, each light distribution having local intensity minima and regions with an intensity increase adjacent thereto, wherein subsequent light distributions are locally modified in accordance with estimated positions of the molecules;
optical means for illuminating two or more spaced-apart molecules in a sample with the light distributions;
a detector unit for detecting photons emitted by the molecules for the different light distributions; and
an apparatus according to claim 18 for determining positions of the two or more molecules in one or more spatial directions.
20. The method according to claim 4 , wherein a second light modulator for influencing the light distributions is arranged in a Fourier plane of the localization microscope, which is arranged to block individual diffraction orders.
21. The method according to claim 4 , wherein the first light modulator is an amplitude modulator or a phase modulator.
22. The method according to claim 5 , wherein for successive position determinations with a light distribution, an intensity profile of the light distribution is changed by changing a structural property of the associated grating structure or ring structure.
23. The method according to claim 4 , wherein the light distributions result from a temporal superposition of different partial light distributions.
24. The method according to claim 4 , wherein a drift is determined from changes in the same direction in the measured positions of molecules in successive measurements.
25. The method according to claim 4 , wherein the photons emitted by the molecules are detected using a camera or an array of photon counters.