FLUORESCENT NANOSCOPY METHOD

US 20120133740A1
Filed: 02/06/2012
Published: 05/31/2012
Est. Priority Date: 05/18/2005
Status: Active Grant

First Claim

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1. A method for 3D microscopy 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 at a plurality of focal planes;

losing the color of the fluorescent agent;

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

determining a light intensity distribution of each of the spots at each of the plurality of focal planes; and

constructing a three-dimensional image of the object using differences between the light intensity distribution for each spot detected between the different focal planes

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Abstract

A method for analysis of an object dyed with fluorescent coloring agents. Separately fluorescing visible molecules or 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 or nanoparticles and for decoloring already recorded fluorescent molecules, wherein tens of thousands of pictures of recorded individual molecule or 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 or nanoparticles. Two-dimensional and three-dimensional images are provided for proteins, nucleic acids and lipids with different coloring agents.

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19 Claims

1. A method for 3D microscopy 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 at a plurality of focal planes;
  
  losing the color of the fluorescent agent;
  
  repeating a cycle of the exciting and registering steps a plurality of times;
  
  determining a light intensity distribution of each of the spots at each of the plurality of focal planes; and
  
  constructing a three-dimensional image of the object using differences between the light intensity distribution for each spot detected between the different focal planes
- View Dependent Claims (2, 3, 4, 5, 6, 7)
- - 2. The method of claim 1, further comprising introducing the fluorescent agent at a low concentration that allows the fluorescent agent to be detectable separately as spots when agent are excited.
  - 3. The method of claim 1, wherein the detector device comprises a light dividing prism and one or more cameras positioned so neighboring focal planes of the object are projected through one object lens.
  - 4. The method of claim 1, further comprising determining lateral coordinates of the spots from the light intensity distributions of the plurality of focal planes.
  - 5. The method of claim 1, wherein the fluorescent agent comprises fluorescent nanoparticles or fluorescent molecules.
  - 6. The method of claim 1, wherein reconstructing the three-dimensional image comprises calculating an axial coordinate of each fluorescent agent spot.
  - 7. The method of claim 1, further comprising determining a difference in light intensity distribution for each spot to calculate a third coordinate of each spot with an accuracy up to several nm, thereby allowing the constructing of the three-dimensional image of the object including a reconstruction of all locations where fluorescing spots 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 the fluorescent agent.

8. A method for 3D microscopy of objects, comprising:
- introducing a fluorescent agent within an object;
  
  exciting the fluorescent agent;
  
  capturing with at least one camera and a data processor a plurality of image frames including separately observed fluorescing spots produced by the excited fluorescent agent;
  
  saving the captured image frames to a recordable medium associated with the data processor;
  
  obtaining more than one intensity distribution for each fluorescing spot from different focal planes with a light dividing prism and the at least one camera; and
  
  constructing a three-dimensional image of the object using differences between the light intensity distributions of each spot detected between the different focal planes.
- View Dependent Claims (9, 10, 11, 12, 13)
- - 9. The method of claim 8, further comprising introducing the fluorescent agent at a low concentration that allows the fluorescent agent to be detectable separately as spots when agent are excited.
  - 10. The method of claim 8, wherein the light dividing prism and one or more cameras are positioned so neighboring focal planes of the object are projected through one object lens.
  - 11. The method of claim 8, wherein the fluorescing spots correspond with lateral coordinates of the fluorescent agent.
  - 12. The method of claim 8, wherein the fluorescent agent comprises fluorescent nanoparticles or fluorescent molecules.
  - 13. The method of claim 8, wherein reconstructing the three-dimensional image comprises calculating an axial coordinate of each spot with an accuracy up to several nm.

14. A method for 3-D microscopy of objects, comprising:
- exciting a fluorescent agent introduced within an object;
  
  registering the excited fluorescent agent as a plurality of separate spots on frames using a detection device;
  
  obtaining different light intensity distributions for each fluorescent agent spot from different focal planes using a light dividing prism and at least one camera positioned so neighboring focal planes of the object project through one object lens;
  
  repeating a cycle of the exciting, registering, and obtaining steps a plurality of times;
  
  calculating a center location of the light intensity distribution of each fluorescent agent spot, wherein the center location corresponds with a lateral coordinate of the fluorescing agent spot; and
  
  creating a three-dimensional image with a data processor from the center locations of the fluorescent agent spots of the repeated cycles.
- View Dependent Claims (15, 16, 17, 18, 19)
- - 15. The method of claim 14, further comprising using differences in the light intensity distributions for each fluorescent agent detected for different focal planes to perform a calculation of an axial coordinate of each fluorescent agent with an accuracy up to several nm.
  - 16. The method of claim 14, wherein the fluorescent agent has a low concentration that allows molecules or nanoparticles of the agent to be detected separately as spots upon the exciting the fluorescent agent.
  - 17. The method of claim 14, wherein the light dividing prism and the at least one camera are positioned so neighboring focal planes of the object are projected through one object lens.
  - 18. The method of claim 14, 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 the separate spot; and
      
      losing the color of the fluorescent molecules.
  - 19. The method of claim 14, 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; and
      
      calculating a location of the nanoparticles from corresponding spots from all frames.

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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

Granted Patent

US 8,334,143 B2
Time in Patent Office

Days
Field of Search
US Class Current

348/46
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

First Claim

1 Assignment

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Abstract

11 Citations

19 Claims

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FLUORESCENT NANOSCOPY METHOD

First Claim

1 Assignment

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0 Petitions

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Accused Products

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Abstract

11 Citations

19 Claims

Specification

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Solutions

Use Cases

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