Fluorescent nanoscopy method

US 8,110,405 B2
Filed: 09/27/2010
Issued: 02/07/2012
Est. Priority Date: 05/18/2005
Status: Expired due to Fees

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1. A method for nanoscopy of objects, comprising:

exciting a fluorescent agent introduced within an object;

registering the excited fluorescent agent as a plurality of separate spots with a detection device;

losing the color of the fluorescent agents;

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

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

creating an image with a data processor from the coordinates of the fluorescent agents registered as spots in the repeated cycles.

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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.

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25 Claims

1. A method for nanoscopy of objects, comprising:
- exciting a fluorescent agent introduced within an object;
  
  registering the excited fluorescent agent as a plurality of separate spots with a detection device;
  
  losing the color of the fluorescent agents;
  
  repeating a cycle of the exciting and registering steps a plurality of times;
  
  using the spots for calculation coordinates of the fluorescent agents; and
  
  creating an image with a data processor from the coordinates of the fluorescent agents registered as spots in the repeated cycles.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
- - 2. A method according to claim 1, wherein the fluorescent agent is a dye and the object is a dyed object, and further 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; and
      
      registering each of the excited fluorescent molecules as a separate spot.
  - 3. A method according to claim 2, 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.
  - 4. A method according to claim 2, 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.
  - 5. A method according to claim 2, further comprisingdyeing 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.
  - 6. A method according to claim 2, wherein the dyed object is placed as a slice of a preserved sample in a polymer.
  - 7. A method according to claim 2, 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.
  - 8. A method according to claim 2, 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;
      
      ordetection of quantities of simultaneously seen and detected separately fluorescing molecules.
  - 9. A method according to claim 2, 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.
  - 10. A method according to claims 2, 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.
  - 11. A method according to claim 2, 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.
  - 12. A method, according to claim 1, further comprising:
    - introducing fluorescing nanoparticles as the fluorescent agent;
      
      registering a motion of the 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.
  - 13. A method according to claim 12, 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.
  - 14. A method according to claim 12, further comprising regulating motion of fluorescent nanoparticles in different directions by electrophoresis with an electric current through two or more electrodes.
  - 15. A method according to claim 12, 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.
  - 16. A method according to claim 12, wherein the nanoparticles comprise phycobiliproteins or fluorescent microspheres.
  - 17. A method according to claim 12, 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.
  - 18. A method according to claim 12, 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;
      
      ordetecting enlarged quantities of simultaneously seen and detected separately fluorescing particles;
      
      orreconstructing local charges of object structures.
  - 19. A method according to claim 12, 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.
  - 20. A method according to claim 1, further comprising:
    - providing a fluorescent microscope equipped with one or more cameras and color-filters for picking up fluorescence light of the 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;
      
      exciting the fluorescent agent with an excitement component for further excitation of newly generated fluorescent molecules and/or fluorescing nanoparticles; and
      
      creating the image using a computer with software 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.
  - 21. A method according to claim 20, 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.
  - 22. A method according to claim 21, wherein the generation component comprises a flash lamp or a UV flash lamp for generation of new fluorescent molecules in an object.
  - 23. A method according to claim 20, 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.
  - 24. A method according to claim 20, 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.
  - 25. A method according to claim 20, further comprising dichroic mirrors and two or more cameras for detection of molecules fluorescing in different wavelengths on different cameras.

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Current Assignee
Super Resolution Technologies LLC (Acacia Research Corporation)
Original Assignee
Stereonic International, Inc.
Inventors
Klimov, Andrey Alexeevich, Klimov, Dmitry Andreevich, Klimov, Evgeniy Andreevich, Klimova, Tatiana Vitalyevna
Primary Examiner(s)
Sines, Brian J

Application Number

US12/891,420
Publication Number

US 20110175982A1
Time in Patent Office

498 Days
Field of Search

436/172, 436/164, 436/174, 977/840, 977/881, 977/868, 977/869, 356/401
US Class Current

436/172
CPC Class Codes

G01N 21/6428   Measuring fluorescence of f...

G01N 21/6458   Fluorescence microscopy flu...

G01N 21/6486   Measuring fluorescence of b...

H04N 13/275   from 3D object models, e.g....

H04N 7/18   Closed-circuit television [...

Y10S 977/84   Manufacture, treatment, or ...

Y10S 977/868   with optical means

Y10S 977/869   Optical microscope

Y10S 977/881   Microscopy or spectroscopy,...

Y10T 436/143333   Saccharide [e.g., DNA, etc.]

Y10T 436/25   including sample preparation

Fluorescent nanoscopy method

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Fluorescent nanoscopy method

First Claim

2 Assignments

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Abstract

25 Citations

25 Claims

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