Sub-diffraction limit image resolution and other imaging techniques

Patent metadata

Field	Value
Publication number	US-10794828-B2
Application number	US-201816058525-A
Country	US
Kind code	B2
Filing date	Aug 8, 2018
Priority date	Aug 7, 2006
Publication date	Oct 6, 2020
Grant date	Oct 6, 2020

Abstract

The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. For example, the entities may be separated by a distance of less than about 1000 nm, or less than about 300 nm for visible light. In one set of embodiments, the entities may be selectively activatable, i.e., one entity can be activated to produce light, without activating other entities. A first entity may be activated and determined (e.g., by determining light emitted by the entity), then a second entity may be activated and determined. The entities may be immobilized relative to each other and/or to a common entity. The emitted light may be used to determine the positions of the first and second entities, for example, using Gaussian fitting or other mathematical techniques, and in some cases, with sub-diffraction limit resolution. The methods may thus be used, for example, to determine the locations of two or more entities immobilized relative to a common entity, for example, a surface, or a biological entity such as DNA, a protein, a cell, a tissue, etc. The entities may also be determined with respect to time, for example, to determine a time-varying reaction. Other aspects of the invention relate to systems for sub-diffraction limit image resolution, computer programs and techniques for sub-diffraction limit image resolution, methods for promoting sub-diffraction limit image resolution, methods for producing photoswitchable entities, and the like.

First claim

What is claimed is:

1. A system, comprising:
an optical system which provides activation light for activating a part of a plurality of photoswitchable entities into a state able to emit light and provides excitation light for exciting at least a part of the activated entities;
a detector which detects at least a part of light emitted from the excited entities; and
a controller, wherein the controller is programmed to calculate positional information of at least a part of the plurality of entities by using information contained in the detected light,
wherein at least some of the entities are cyanine dyes.

2. The system of claim 1 , wherein the optical system comprises a first optical system which provides activation light and a second optical system provides excitation light.

3. The system of claim 2 , wherein the activation light and the excitation light have substantially the same wavelength.

4. The system of claim 2 , wherein the activation light and the excitation light have different wavelengths.

5. The system of claim 1 , wherein the cyanine dyes comprise at least one of Cy5, Cy5.5, Cy7, Alexa Fluor® 647, Alexa Fluor® 405, Alexa Fluor® 488, Cy2, Cy3, Cy3.5, Cy5, and a conjugate thereof.

6. The system of claim 1 , wherein the controller is programmed to correct the positional information by using a fiduciary marker and movements of the fiduciary marker.

7. The system of claim 6 , wherein the correction of the positional information comprises drift correction.

8. The system of claim 6 , wherein the fiduciary marker is a fluorescent particle.

9. The system of claim 1 , wherein the controller is able to use Gaussian fitting to calculate the positional information.

10. The system of claim 1 , wherein the controller is able to use elliptical Gaussian fitting to calculate the positional information.

11. The system of claim 1 , wherein the controller is able to generate an image by using the calculated positional information.

12. The system of claim 11 , wherein the image is a high resolution image.

13. The system of claim 1 , wherein the plurality of entities comprises a first entity and a second entity.

14. The system of claim 13 , wherein:
the first entity is activatable by a first wavelength of activation light and
the second entity is activatable by a second wavelength of activation light.

15. The system of claim 13 , wherein:
the first entity is excitable by a first wavelength of excitation light and
the second entity is excitable by a second wavelength of excitation light.

16. The system of claim 1 , wherein the entities have a first portion and a second portion, the first portion being a light emitting portion and the second portion being an activator portion.

17. The system of claim 1 , wherein the controller is able to calculate positional information of at least some of the plurality of entities to a resolution smaller than 20 nm.

18. The system of claim 1 , wherein the controller is able to use the positional information to construct an image, the image having a resolution that is better than the diffraction-limited resolution of the emitted light.

19. The system of claim 1 , wherein the controller is able to calculate the positional information of the at least a part of the plurality of entities at more than one point of time and/or as a function of time.

20. The system of claim 1 , wherein at least some of the plurality of entities are activatable by light of different wavelengths and/or emit light at different wavelengths.

21. A program for causing a machine to perform a procedure of calculating positional information performed by a system as claimed in claim 1 .

22. A storage medium comprising a program for causing a machine to perform a procedure of calculating positional information performed by a system as claimed in claim 1 .

23. An article, comprising a storage medium comprising a program for causing a machine to perform a procedure of calculating positional information performed by a system as claimed in claim 1 .

24. A method, comprising:
applying activation light for activating a part of a plurality of photoswitchable entities into a state able to emit light, wherein the entities are cyanine dyes;
applying excitation light for exciting at least a part of the activated entities;
detecting at least a part of light emitted from the excited entities; and
calculating positional information of at least a part of the plurality of entities by using information contained in the detected light.

Assignees

• Harvard College

Inventors

• Zhuang Xiaowei
• Bates Wilfred M
• Rust Michael J

Classifications

• G01N21/6458

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

• G01N21/6428 Primary

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N21/6408

  with measurement of decay time, time resolved fluorescence · CPC title

• G01N33/582

  with fluorescent label · CPC title

• G02B27/58

  Optics for apodization or superresolution; Optical synthetic aperture systems · CPC title