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Single‐pinhole confocal imaging of sub‐resolution sparse objects using experimental point spread function and image restoration
Author(s) -
Diaspro A.,
Annunziata S.,
Robello M.
Publication year - 2000
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/1097-0029(20001201)51:5<464::aid-jemt9>3.0.co;2-d
Subject(s) - pinhole (optics) , point spread function , optics , confocal , full width at half maximum , resolution (logic) , physics , point (geometry) , confocal microscopy , materials science , microscope , image resolution , wavelength , optical sectioning , computer science , artificial intelligence , mathematics , geometry
Sparse fluorescent pointlike subresolution objects have been imaged using a diffraction limited single‐pinhole confocal fluorescence microscope. A Maximum likelihood image restoration algorithm has been used in conjunction with a measure of the experimental point spread function for improving the three‐dimensional imaging of subresolution sparse objects. The experimental point‐spread‐function profiles have been improved by a factor of 1.95 in lateral direction and 3.75 in axial direction resulting in full‐width half maximum (FWHM) values of 91 ± 11 nm and 160 ± 26 nm. This amounts to 1.43 and 2.15 in optical units, respectively. The lateral and axial FWHM of the sparse pointlike subresolution objects is about 5 and 3 times smaller than the wavelength. This result points to the attractive possibility of utilising a compact confocal architecture for localising punctuate fluorescent objects having subresolution dimensions. The key resides in the utilisation of the measured point spread function coupled to an appropriate image restoration approach, and, of course, in the stability of the confocal system being used. Microsc. Res. Tech. 51:464–468, 2000. © 2000 Wiley‐Liss, Inc.