Devices, methods, and systems relating to super resolution imaging

Patent metadata

Field	Value
Publication number	US-10830639-B2
Application number	US-201515514084-A
Country	US
Kind code	B2
Filing date	Sep 25, 2015
Priority date	Sep 25, 2014
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

Certain examples disclose systems and methods for imaging a target. An example method includes: a) activating a subset of light-emitting molecules in a wide field area of a target using an excitation light; b) capturing one or more images of the light emitted from the subset of the molecules illuminated with the excitation light; c) localizing one or more activated light emitting molecules using one or more single molecule microscopic methods to obtain localization information; d) simultaneously capturing spectral information for the same localized activated light emitting molecules using one or more spectroscopic methods; e) resolving one or more non-diffraction limited images of the area of the target using a combination of the localization and spectral information for the localized activated light emitting molecules; and f) displaying the one or more non-diffraction limited images.

First claim

What is claimed is:

1. A method for imaging a target, the method comprising:
(a). activating a subset of light-emitting molecules in a wide-field area of the target using an excitation light;
(b). capturing one or more images of the light emitted from the subset of the molecules illuminated with the excitation light;
(c). dividing light emitted from the subset of the molecules illuminated with the excitation light into a first light and a second light;
(d). adjusting a position of the first light on an imager via a mirror, wherein the mirror is positioned such that the first light but not the second light is reflected by said mirror;
(e). using the first light, localizing one or more activated light emitting molecules, using one or more single molecule microscopic methods to obtain localization information; wherein the step of localizing comprises summing the localization information from the same one or more activated light emitting molecules to improve a localization precision;
(f). simultaneously, using the second light, capturing spectral information for the same localized activated light emitting molecules using one or more spectroscopic methods, such that the localization information is obtained and the spectral information is captured simultaneously for each of the one or more activated light emitting molecules;
(g). resolving one or more non-diffraction limited images of the wide-field area of the target from the one or more captured images using a combination of the localization and spectral information for the localized activated light emitting molecules; and
(h). displaying the one or more non-diffraction limited images.

2. The method of claim 1 , wherein the step of activating the subset of molecules further comprises illuminating the sample with low-intensity activation light that stochastically photoswitches the subset of light-emitting particles.

3. The method of claim 1 , wherein the light-emitting molecules are not extrinsically labeled.

4. The method of claim 1 , wherein the light-emitting molecules comprises one or more light-emitting extrinsic labels.

5. The method of claim 4 , wherein the light-emitting extrinsic label is selected from the group consisting of fluorescent tag, fluorescent protein, fluorophore, fluorescent probe, quantum dot, fluorescence resonance energy transfer probe, and diode laser excitable probe.

6. The method of claim 1 , wherein the step of resolving one or more non-diffraction limited images further comprises capturing one or more zero-order images and one or more first-order spectral images simultaneously or substantially simultaneously.

7. The method of claim 1 , wherein the one or more single molecule microscopic methods is selected from the group consisting of stochastic optical reconstruction microscopy, spectral precision distance microscopy (SPDM), spectral precision distance microscopy with physically modifiable fluorophores (SPDMphymod), photo activated localization microscopy (PALM), photo-activation localization microscopy (FPALM), photon localization microscopy (PLM), direct stochastical optical reconstruction microscopy (dSTORM), super-resolution optical fluctuation imaging (SOFI), and 3D light microscopical nanosizing microscopy (LIMON).

8. The method of claim 7 , wherein the step of resolving one or more non-diffraction limited images further comprises determining the centroid positions of the one or more localized activated light-emitting molecules and establishing a reference point of the corresponding emission spectrum of one or more localized activated light-emitting molecules.

9. The method of claim 1 , wherein the step of resolving one or more non-diffraction limited images further comprises resolving individual spectral curves with a spectral linear unmixing algorithm and analysis by spectral regression.

10. The method of claim 1 , wherein the step of resolving one or more non-diffraction limited images further comprises normalizing spectra.

11. The method of claim 1 , wherein the emission spectra from the same activated light-emitting molecule are classified by spectral regression.

12. The method of claim 1 , wherein the one or more spectroscopic methods is selected from the group consisting of Raman spectroscopy, optical fluorescence microscopy, infrared spectroscopy, ultraviolet spectroscopy, laser microscopy and confocal microscopy.

13. The method of claim 1 , wherein the step of activating a subset of light-emitting molecules in the wide-field area of the target and the step of capturing one or more images of the light emitted from the subset of the particles illuminated with excitation light is performed with one or more light wavelengths selected outside of the primary absorption-emission bands of the target.

14. The method of claim 1 , wherein the target is selected from the group consisting of polymer, protein, nucleic acid, lipid, carbohydrate, cell, cells, subcellular organelles, subcellular structures, extracellular structures, nanofabricated structures, nanoparticles, nanostructures, semiconductor chips, and crystals.

15. The method of claim 1 , wherein the step of resolving one or more non-diffraction limited images further comprises applying an agent to the target that reduces photobleaching.

16. The method of claim 1 , wherein the step of resolving one or more non-diffraction limited images is performed in three dimensions (3D).

17. The method of claim 1 , wherein the step of resolving one or more non-diffraction limited images further comprises summing the resolved images.

18. The method of claim 1 , wherein the step of capturing one or more images of the light emitted from the subset of the particles illuminated with excitation light is performed using intrinsic contrast of the target.

19. The method of claim 1 , wherein the step of displaying the one or more non-diffraction limited images is used to detect a feature of the target selected from the group consisting of structural feature, chemical feature, structural defect, chemical defect, target quality, disease, disease state, target sequence and target composition.

20. The method of claim 1 , wherein the step of resolving one or more non-diffraction limited images is performed with an imaging resolution less than 100 nm.

21. A system for imaging a target, the system comprising:
(a). one or more light sources configured to activate a subset of light-emitting molecules, in a wide field area of the target;
(b). a microscope feature configured to capture optical images of the subset of light-emitting molecules in the wide field area of the target;
(c). a spectrometer configured to simultaneously capture spectral information for individual light-emitting molecules in the subset of light-emitting molecules in the wide field area of the target while localizing the one or more activated light emitting molecules to obtain localization information; such that the localization information is obtained and the spectral information is captured simultaneously for each of the activated light emitting molecules;
(d). a spectral filtering element;
(e). one or more imagers configured to process the optical images, localization information, and spectral information for light-emitting molecules in the wide field area of the target to generate one or more non-diffraction limited images of the wide field area;
(f). a display for one or more non-diffraction limited images of the wide field area;
wherein the system is configured to divide light emitted from the subset of the molecu

Assignees

• Univ Northwestern

Inventors

• Urban Ben
• Zhang Hao F
• Sun Cheng
• Dong Biqin

Classifications

• G02B21/0076 Primary

  arrangements using fluorescence or luminescence · CPC title

• G02B21/0064

  multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling (G02B21/0076 takes precedence) · CPC title

• G01N21/6458

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

• G02B21/008

  Details of detection or image processing, including general computer control · CPC title

• G02B21/0032

  Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers (G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06) · CPC title