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Localization of fluorescent particles with nanometer precision using standard microscopy and a point spread function approach
Author(s) -
Marki Alex,
Ermilov Eugeny,
Secomb Timothy W.,
Pries Axel R.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.683.5
Subject(s) - standard deviation , point spread function , microscopy , optics , microscope , particle (ecology) , optical microscope , fluorescence , radius , materials science , resolution (logic) , nanometre , range (aeronautics) , fluorescence microscope , physics , analytical chemistry (journal) , chemistry , mathematics , scanning electron microscope , statistics , computer science , chromatography , oceanography , computer security , artificial intelligence , composite material , geology
Background Resolution of optical microscopy is limited by wave optics to about 200 to 400 nm. However, diffraction patterns generated by individual fluorescent particles (point spread functions, PSF) contain location information with much higher precision. Here, we describe a user‐friendly approach for single fluorescence particle 3D localization and tracking with nanometer precision in a standard fluorescence microscope. Methods Images of fluorescent particles were obtained over a large range (about 7 μm) of vertical positions and analysed by whole pattern (WP) comparison with a PSF recorded for the specific setup and measurement of the outermost ring radius (ORR). Results For a vertical range of 4.5 μm, standard deviation (SD) from the predicted value, indicating validity, was 15.6 and 23.5 nm for WP and ORR. Underexposure or overlapping images led only to a slight increase in SD. The precision, determined as SD of the distance between two particles, ranged from 3 to 6 nm. Local viscosity may be determined from random particle movement with a precision of about 5% of the measured value. Conclusion With a validity in the range of 20 nm and a precision of about 4–5 nm obtained by standard fluorescent microscopy, the PSF approach offers a valuable tool for a variety of experimental investigations of particle/structure localizations and local viscosity, e.g. in studies of the endothelial surface layer.