Spectrally resolved super-resolution microscopy and ultrahigh-throughput single-molecule spectroscopy

1. A system for spectrally resolved super-resolution microscopy, comprising:

(a) a computer processor; and

(b) a non-transitory computer-readable memory storing instructions executable by the computer processor;

(c) wherein said instructions, when executed by the computer processor, perform steps comprising:

(i) illuminating a sample comprising a plurality of molecules;

(ii) generating a non-dispersed wide-field image of a molecule within the sample; and

(iii) simultaneously dispersing fluorescence of the molecule to concurrently acquire spectral data and location data of the molecule in wide-field within the image.

2. The system of claim 1 , wherein illuminating the sample comprises stochastic switching of the molecules from a non-emitting dark state and an emitting fluorescent state for synchronous spectrum measurement and localization of the plurality of molecules.

3. The system of claim 2 :

wherein illuminating the sample comprises illuminating the sample such that a first subset of the molecules are placed into the non-emitting dark state and a smaller, sparsely-distributed second subset of the molecules are placed into the emitting fluorescent state such that the fluorescence of the second subset of emitting molecules is dispersed into non-overlapping spectra in wide-field.

4. The system of claim 2 , wherein the spectral data and location data are acquired at a throughput rate of at least ten thousand of molecules per second.

5. The system of claim 1 , said instructions when executed by the computer processor further perform steps comprising:

constructing a spectrally-resolved, stochastic optical reconstruction microscopy (SR-STORM) super-resolution image of the sample from a plurality of single-molecule images and localized positions of single molecules.

6. The system of claim 1 , said instructions when executed by the computer processor further perform steps comprising:

simultaneously imaging the sample in a first path and a second path;

wherein the first path is configured to generate a first image comprising the non-dispersed wide-field image of the molecule; and

wherein the second path is configured to generate a second image comprising the dispersed spectral data of the molecule in wide-field.

7. The system of claim 6 , said instructions when executed by the computer processor further perform steps comprising:

simultaneously detecting the first image and second image on a single wide-field image frame.

8. The system of claim 6 , said instructions when executed by the computer processor further perform steps comprising:

mapping localized positions of single molecules acquired from the first path to spectrum data acquired from the second path.

9. The system of claim 8 , said instructions when executed by the computer processor further perform steps comprising:

calculating spectral positions of different wavelengths for each molecule.

10. The system of claim 8 , said instructions when executed by the computer processor further perform steps comprising:

generating a calibrated spectrum for each molecule.

11. The system of claim 6 , further comprising:

at least one optical element directed at the sample and disposed within the first path and the second path for generating the first and second images; and

a detector disposed within the first path and the second path for receiving the first and second images.

12. The system of claim 11 , wherein the at least one optical element comprises:

a first objective; and

a beam splitter in an optical path the first objective for splitting the optical path into the first path and the second path.

13. The system of claim 11 , wherein the at least one optical element comprises:

a first objective and a second objective at opposing ends of the sample; and

wherein the first objective is configured to generate the first path and the second objective is configured to generate the second path.

14. The system of claim 11 , further comprising an illumination source for illuminating the sample;

wherein illuminating the sample comprises stochastic switching of the molecules from a non-emitting dark state and an emitting fluorescent state for synchronous spectrum measurement and localization of the plurality of molecules.

15. The system of claim 14 :

wherein the detector comprises a camera having a frame rate; and

wherein stochastic switching of the molecules from a non-emitting dark state and an emitting fluorescent state is switched off at a rate configured to substantially match the frame rate of the camera.

16. The system of claim 15 , wherein stochastic switching of the molecules from a non-emitting dark state and an emitting fluorescent state is switched on at a rate configured to achieve an optimal density of emitting molecules.

17. The system of claim 11 , wherein the second path comprises:

an image mode and a spectrum mode; and

a stage disposed within the second path to switch from the image mode to the spectrum mode;

wherein said instructions are further configured for controlling translation of the stage from the image mode to the spectrum mode.

18. The system of claim 17 :

the stage further comprising a prism and two relay lenses within the second optical path; and

wherein prism is positioned in a Fourier plane of the two relay lenses for dispersing spectra within the second image in the spectrum mode.

19. A method for spectrally resolved super-resolution microscopy, comprising:

illuminating a sample comprising a plurality of molecules;

generating a non-dispersed wide-field image of a molecule within the sample; and

simultaneously dispersing fluorescence of the molecule to concurrently acquire spectral data and location data of the molecule in wide-field within the image.

20. The method of claim 19 , wherein illuminating the sample comprises stochastic switching of the molecules from a non-emitting dark state and an emitting fluorescent state for synchronous spectrum measurement and localization of the plurality of molecules.

21. The method of claim 20 :

wherein illuminating the sample comprises illuminating the sample such that a first subset of the molecules are placed into the non-emitting dark state and a smaller, sparsely-distributed second subset of the molecules are placed into the emitting fluorescent state such that the fluorescence of the second subset of emitting molecules is dispersed into non-overlapping spectra in wide-field.

22. The method of claim 20 , wherein the spectral data and location data are acquired at a throughput rate of at least ten thousand of molecules per second.

23. The method of claim 19 , further comprising:

constructing a spectrally-resolved, stochastic optical reconstruction microscopy (SR-STORM) super-resolution image of the sample from a plurality of single-molecule images and localized positions of single molecules.

24. The method of claim 19 , further comprising:

simultaneously imaging the sample in a first path and a second path;

wherein the first path is configured to generate a first image comprising the non-dispersed wide-field image of the molecule; and

wherein the second path is configured to generate a second image comprising the dispersed spectral data of the molecule in wide-field.

25. The method of claim 24 , further comprising:

simultaneously detecting the first image and second image on a single wide-field image frame.

26. The method of claim 24 , further comprising:

mapping localized positions of single molecules acquired from the first path to spectrum data acquired from the second path.

27. The method of claim 26 , further comprising:

calculating spectral positions of different wavelengths for each molecule.

28. The method of claim 26 , further comprising:

generating a calibrated spectrum for each molecule.

29. The method of claim 24 :

wherein imaging of the sample is acquired with a camera having a frame rate;

wherein illuminating the sample comprises stochastic switching of the molecules from a non-emitting dark state and an emitting fluorescent state for synchronous spectrum measurement and localization of the plurality of molecules; and

wherein stochastic switching of the molecules from a non-emitting dark state and an emitting fluorescent state is switched off at a rate configured to substantially match the frame rate of the camera.

30. The method of claim 29 , wherein stochastic switching of the molecules from a non-emitting dark state and an emitting fluorescent state is switched on at a rate configured to achieve an optimal density of emitting molecules.

31. The method of claim 24 , wherein the second path comprises an image mode and a spectrum mode for dispersing spectra within the second image, the method further comprising:

switching the second path from the image mode to the spectrum mode.

32. An apparatus for spectrally resolved super-resolution microscopy, comprising:

at least one optical element directed at a sample comprising a plurality of molecules;

an illumination source configured for illuminating the sample;

wherein the at least one optical element is configured to comprise a first path and a second path;

wherein the first path is configured to generate a first image comprising a non-dispersed wide-field image of a molecule within the sample; and

wherein the second path is configured to concurrently generate a second image comprising dispersed spectral data of the molecule in wide-field; and

a detector coupled to the first path and the second path for receiving the first and second images to concurrently acquire spectral data and location data of the molecule in wide-field within the image.

33. The apparatus of claim 32 , wherein the at least one optical element comprises:

a first objective; and

a beam splitter in an optical path the first objective for splitting the optical path into the first path and the second path.

34. The apparatus of claim 32 , wherein the at least one optical element comprises:

a first objective and a second objective at opposing ends of the sample; and

wherein the first objective is configured to generate the first path and the second objective is configured to generate the second path.

35. The apparatus of claim 32 , further comprising an illumination source for illuminating the sample;

wherein the illumination source is configured to illuminate the sample via stochastic switching of the molecules from a non-emitting dark state and an emitting fluorescent state for synchronous spectrum measurement and localization of the plurality of molecules.

36. The apparatus of claim 35 :

wherein the detector comprises a camera having a frame rate; and

wherein stochastic switching of the molecules from a non-emitting dark state and an emitting fluorescent state is switched off at a rate configured to substantially match the frame rate of the camera.

37. The apparatus of claim 32 , wherein the second path comprises:

an image mode and a spectrum mode; and

a stage disposed within the second path to switch from the image mode to the spectrum mode;

wherein said instructions are further configured for controlling translation of the stage from the image mode to the spectrum mode.

38. The apparatus of claim 37 :

the stage further comprising a prism and two relay lenses within the second optical path; and

wherein prism is positioned in a Fourier plane of the two relay lenses for dispersing spectra within the second image in the spectrum mode.