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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.

Original publication

DOI

10.1111/j.1365-2818.2011.03544.x

Type

Journal article

Journal

J Microsc

Publication Date

01/2012

Volume

245

Pages

63 - 71