Resolution in optical microscopy

Titelangaben

Jonkman, James E. N. ; Swoger, Jim ; Kress, Holger ; Rohrbach, Alexander ; Stelzer, Ernst H. K.:
Resolution in optical microscopy.
In: Marriott, Gerard ; Parker, Ian (Hrsg.): Biophotonics. Part A. - Amsterdam : Academic Press , 2003 . - S. 416-446 . - (Methods in Enzymology ; 360 )
ISBN 0-12-182263-X
DOI: https://doi.org/10.1016/S0076-6879(03)60122-9

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Abstract

Publisher Summary This chapter describes resolution in optical microscopy. Optical microscopes are fundamentally limited in the resolution they can achieve. The resolution depends on the wavelength of the light (both incident and detected), on the numerical aperture (NA) of the optical arrangement, and on the specimen to be observed or the experiment to be performed. Live specimens are also dynamic and sensitive to photobleaching and thermal damage, which imposes a limit on the duration for which they can be observed and on the power of the incident light. Fluorescence is excited throughout its illumination cone, but only fluorescence emitted from the focal point is imaged through the confocal pinhole to the detector. A useful tool for comparing the performance of optical microscopes is the point-spread function (PSF). The PSF can be defined in two complementary. The intensity PSF (hereafter referred to simply as the PSF) can be measured by taking images of—for example, a subresolution bead as it is scanned through the focus of a microscope. Real specimens are, of course, rarely point sources.