METHOD OF FLUORESCENT NANOSCOPY

• US 20110175982A1
• Filed: 09/27/2010
• Published: 07/21/2011
• Est. Priority Date: 05/18/2005
• Status: Active Grant

• Current Assignee: Super Resolution Technologies LLC (Acacia Research Corporation)
• Original Assignee: Stereonic International, Inc.
• Inventors: Klimov, Andrey Alexeevich, Klimova, Tatiana Vitalyevna, Klimov, Dmitry Andreevich, Klimov, Evgeniy Andreevich
• Granted Patent: US 8,110,405 B2
• Time in Patent Office: Days
• US Class Current: 348/46
• CPC Class Codes: G01N 21/6428 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 Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: H04N 13/275 from 3D object models, e.g. computer-generated stereoscopic image signals
• CPC Class Codes: H04N 7/18 Closed-circuit television [CCTV] systems, i.e. systems in which the video signal is not broadcast
• CPC Class Codes: Y10S 977/84 Manufacture, treatment, or detection of nanostructure
• CPC Class Codes: Y10S 977/868 with optical means
• CPC Class Codes: Y10S 977/869 Optical microscope
• CPC Class Codes: Y10S 977/881 Microscopy or spectroscopy, e.g. sem, tem
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

An analysis of an object dyed with fluorescent coloring agents carried out with the aid of a fluorescent microscope which is modified for improved resolving power and called a nanoscope. The method is carried out with a microscope having an optical system for visualizing and projecting a sample image to a video camera which records and digitizes images of individual fluorescence molecules and nanoparticles at a low noise, a computer for recording and processing images, a sample holder arranged in front of an object lens, a fluorescent radiation exciting source and a set of replaceable suppression filters for separating the sample fluorescent light. Separately fluorescing visible molecules and nanoparticles are periodically formed in different object parts, the laser produces the oscillation thereof which is sufficient for recording the non-overlapping images of the molecules and nanoparticles and for decoloring already recorded fluorescent molecules, wherein tens of thousands of pictures of recorded individual molecule and nanoparticle images, in the form of stains having a diameter on the order of a fluorescent light wavelength multiplied by a microscope amplification, are processed by a computer for searching the coordinates of the stain centers and building the object image according to millions of calculated stain center co-ordinates corresponding to the co-ordinates of the individual fluorescent molecules and nanoparticles. With this invention it is possible to obtain a two-dimensional and a three-dimensional image with a resolving power better than 20 nm and to record a color image by dyeing proteins, nucleic acids and lipids with different coloring agents.

Claims

1-5. -5. (canceled)

6. A method for nanoscopy of dyed objects, comprising:

generating fluorescent molecules by converting non-fluorescent dye molecules into fluorescent dye molecules;

exciting the fluorescent molecules of the dyed object, wherein each of the excited fluorescent molecules has a color;

registering each of the excited fluorescent molecules as a separate spot;

losing the color of the fluorescent molecules;

repeating a cycle of the exciting and registering steps a plurality of times;

using the spots for calculation coordinates of the fluorescent molecules; and

creating an image from the coordinates of the fluorescent molecules registered as spots in the repeated cycles.

7. A method according to claim 6, further comprising:

detecting the excited fluorescent molecules using a fluorescent microscope equipped with one or more cameras and color-filters for picking up fluorescence light of the excited dyed object, the one or more cameras being able to detect low noise level frames with images of separate fluorescing fluorescent molecules which are excited by a laser, the microscope being also equipped with recordable medium for recording images received from the cameras;

generating fluorescent molecules by converting non-fluorescent dye molecules in a field of view of the one or more cameras into fluorescent dye molecules by chemical reaction or physical influence;

registering each of the excited fluorescent molecules as separate spots on video frames using the one or more cameras;

saving sequential frames with images of newly formed fluorescent molecules on the recordable medium;

repeating the cycle a plurality of times; and

calculating locations of molecules from spot centers and distributions of intensity received for images of separate fluorescent molecules using the saved frames, the locations or distributions having different locations on sequential frames, the locations being correspondent with coordinates of fluorescing molecules, wherein the calculation of locations of fluorescing molecules provides a resolution comparable with sizes of the fluorescing molecules, up to several nm;

wherein the process provides a resolution of molecular structures for layers of the object which are located on the distance not more than 150 nm from glass and provides resolution for larger distances depending from mobility of object structures.

8. A method according to claim 6, wherein the fluorescent molecules lose color after registration of their fluorescence either as spots with intensity which allows calculation of locations of fluorescing molecules or on each frame.

9. A method according to claim 6, further comprising

dying an object with a saturating concentration of a dye, placed in a solution, to provide the dyed object;

washing away superfluous dye; and

sealing gaps on edges of glasses holding the dyed object to avoid drying of the dye solution.

10. A method according to claim 6, wherein the dyed object is placed as a slice of a preserved sample in a polymer.

11. A method according to claim 6, further comprising:

saving frames with residual fluorescence and frames containing images of fluorescing molecules overlapped on residual fluorescence to a recordable medium;

subtracting frames with residual fluorescence from frames containing images of fluorescing molecules overlapped on residual fluorescence; and

using subtracted frames for calculation of a location of the fluorescing molecules based on a calculation of coordinates of spot centers and distribution of intensity received for images of each fluorescent molecule.

12. A method according to claim 6, further comprising:

using different dyes for dyeing different structures of the object into different colors, wherein the different dyes including dyes fluorescing at different wavelengths or having different active groups, wherein the different active groups are active groups suited to link to proteins, active groups suited to link to nucleic acids, or active groups suited to link to fats;

using dichroic mirrors for detection of molecules fluorescing in different wavelengths;

using dichroic mirrors and two or more cameras for detection of molecules fluorescing in different wavelengths to allow reconstruction of locations of several different types of object molecules;

or

detection of quantities of simultaneously seen and detected separately fluorescing molecules.

13. A method according to claim 6, further comprising:

periodically converting non-fluorescing dye molecules into fluorescing dye molecules by a flash lamp, UV flash lamp, UV pulse light source, ATP reactions, esterase influence, peroxidation, or radioactive transformation.

14. A method according to claim 6, further comprising:

detecting key particles in the dyed object using normal fluorescence microscopy or using frames with residual fluorescence, wherein the key particles are bright shining fluorescing particles, particles rigidly connected with the object, particles introduced into the dyed object; and

calculating the coordinates of the fluorescent molecules using at least some of the key particles within the object, taking into account movement in a vision area of the key particles for receiving a resolution up to several nm even for objects with some mobility of object structures.

15. A method according to claim 6, further comprising:

using a light dividing prism and two cameras that are located so that two neighboring focal planes of the object are projected through one object lens, having a light intensity distribution for each fluorescent molecule be different when detected by different cameras, using the said difference of intensity for calculation of the third coordinate to create a 3-D image of the object.

16. A method for nanoscopy of objects, comprising:

registering a motion of fluorescing nanoparticles resistant to fluorescent color loss within a portion of a sample filled with solution, the motion allowing a scan of accessible volume of the sample and to indicate volume that is filled with liquid and volume that is occupied with dense structures;

calculating a location of the nanoparticles from corresponding spots from all frames.

17. A method according to claim 16, wherein the method is used for 2D nanoscopy or 3D nanoscopy of the objects being placed in a solution, and uses a fluorescent microscope, a laser, light dividing prisms and color filters, one or more cameras for detecting fluorescence light of the object that are able to detect images of separate fluorescing nanoparticles that are moveable between structures of the object and excited by a laser, and a computer for recording images received from cameras, the method comprising:

exciting the fluorescence of the fluorescent nanoparticles;

detecting with the cameras and the computer thousands of frames with separately observed spots, each of the spots reflecting a location of a fluorescing nanoparticles;

projecting an image of the object on cameras through one object lens and light dividing prisms;

using saved frames to calculate locations of centers of distribution of intensity received for images of each fluorescent nanoparticle on the saved frames, wherein having different locations on sequential frames, the locations correspond with coordinates of fluorescing nanoparticles; and

using differences in light intensity distribution for each fluorescent nanoparticle detected by different cameras to perform a calculation of a third coordinate of each fluorescent nanoparticle with an accuracy up to several nm, thereby allowing reconstructing a 3D image of the object including a reconstruction of all locations where fluorescing nanoparticles appeared over a time period which indicates a volume that is occupied with liquid and the volume where particles were not able to be present, which can be considered to be occupied with dense structures or structures having the same local charge as nanoparticles;

wherein the process features a stability for resolution of molecular structures for layers of the object which are located on a distance not more than 150 nm from glass and provides resolution for larger distances depending from mobility of object structures.

18. A method according to claim 16, further comprising regulating motion of fluorescent nanoparticles in different directions by electrophoresis with an electric current through two or more electrodes.

19. A method according to claim 16, further comprising:

studying surface charges of object structures by simultaneously using fluorescent nanoparticles with positive charges, nanoparticles with negative charges, and neutral nanoparticles which interact with local charges of structures and move between different areas of the object.

20. A method according to claim 16, wherein the nanoparticles comprise phycobiliproteins or fluorescent microspheres.

21. A method according to claim 16, further comprising:

detecting key particles using normal fluorescence microscopy or using frames with residual fluorescence, wherein the key particles are bright shining fluorescing particles, particles rigidly connected with the object, or particles introduced into object; and

calculating coordinates of the fluorescent nanoparticles using at least some of the key particles within the object, taking into account movement in a vision area of the key particles for receiving resolution up to several nm even for structures of the object having mobility.

22. A method according to claim 16, further comprising:

detecting nanoparticles fluorescing at more than one wavelength using dichroic mirrors and two or more cameras; and

reconstructing a location of several different types of fluorescing particles in the object;

or

detecting enlarged quantities of simultaneously seen and detected separately fluorescing particles;

or

reconstructing local charges of object structures.

23. A method according to claim 16, further comprising:

using a light dividing prism and cameras that are located so that two neighboring focal planes of the object are projected through one object lens;

obtaining two different light intensity distributions for each fluorescent particle, each detected by one of the cameras; and

using a difference between the two intensity distributions for calculation of a third coordinate to create a 3-D image of the object.

24. A fluorescent microscope-nanoscope device comprising:

a fluorescent microscope equipped with one or more cameras and color-filters for picking up fluorescence light of the dyed object, such cameras being able to detect, with a low noise level, frames with images of separate excited fluorescing nanoparticles, moving between structures of object, and/or fluorescing molecules rigidly connected with object structures;

an excitement component for further excitation of newly generated fluorescent molecules and/or fluorescing nanoparticles; and

a computer with software used for synchronization of different types of illumination with time of frame capture, for calculation of coordinates of fluorescent molecules and nanoparticles and for creation of 2-D or 3-D images of the object.

25. A device according to claim 24, further comprising a generation component that periodically turns at least hundreds of non-fluorescent dye molecules in a field of view into fluorescent dye molecules by a chemical reactions or a physical influence.

26. A device according to claim 24, wherein the generation component comprises a flash lamp or a UV flash lamp for generation of new fluorescent molecules in an object.

27. A device according to claim 24, further comprising:

an electrophoresis device for electrophoresis of fluorescing nanoparticles in different directions with current, including two or more electrodes and the microscope further comprising a recordable medium for recording images received from the cameras.

28. A device according to claim 24, further comprising:

a light dividing prism and two cameras that are located so that two neighboring focal planes of the object are projected on the two cameras through one object lens, wherein each of the two cameras detects a different light intensity distribution for each fluorescent particle and/or molecule; and

software for calculating a third coordinate using the difference between the intensity distributions detected by the two cameras, and for creating a 3-D image of the object.

29. A device according to claim 24, further comprising dichroic mirrors and two or more cameras for detection of molecules fluorescing in different wavelengths on different cameras.

30. A device according to claim 24, further comprising a laser for exciting fluorescence in the object through an objective lens, through an object glass, on a full depth, or under an angle of total internal reflection.