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Effects of aberrations and specimen structure in conventional, confocal and two‐photon fluorescence microscopy
Author(s) -
SIMMONDS R. D.,
WILSON T.,
BOOTH M. J.
Publication year - 2012
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 - 10.1111/j.1365-2818.2011.03544.x
Subject(s) - confocal microscopy , fluorescence , two photon excitation microscopy , microscopy , confocal , optics , fluorescence microscope , materials science , multiphoton fluorescence microscope , biophysics , chemistry , physics , biology
Summary Specimen‐induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as point‐like, linear, planar and volume structures, when imaged by conventional, confocal and two‐photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two‐photon microscopes over conventional instruments when aberrations are present.