Premium
Contrast, resolution, pixelation, dynamic range and signal‐to‐noise ratio: fundamental limits to resolution in fluorescence light microscopy
Author(s) -
Stelzer
Publication year - 1998
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1046/j.1365-2818.1998.00290.x
Subject(s) - optics , resolution (logic) , microscopy , light sheet fluorescence microscopy , microscope , numerical aperture , image resolution , confocal , materials science , dynamic range , contrast (vision) , confocal microscopy , fluorescence , aperture (computer memory) , wavelength , physics , temporal resolution , optical microscope , scanning confocal electron microscopy , computer science , artificial intelligence , acoustics , scanning electron microscope
In a perfect optical system numerical aperture and wavelength determine resolution. In a real optical system, however, the number of photons collected from a specimen determines the contrast and this limits the resolution. Contrast is affected by the number of picture elements per unit area, the number of photons and the aberrations present in every optical system. The concept of contrast vs. distance functions is used to compare the resolution achievable in confocal and wide‐field fluorescence microscopes and the effect of a further reduction of the observable volume. In conclusio : (a) real optical systems will never be able to achieve the theoretical resolution, (b) wide‐field fluorescence microscopy will often provide a better resolution than confocal fluorescence microscopy, (c) decreasing the observed volume does not necessarily increase the resolution and (d) using multiple fluorophores can improve the accuracy with which distances are measured. Some numbers for typical situations are provided.