Fluorescent nanoscopy method
DCFirst Claim
1. 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.
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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.
27 Citations
12 Claims
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1. A method for 3D microscopy of objects, comprising:
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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 (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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Specification