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Three dimensional (3-D) fluorescence microscopes, including conventional instruments with digital deblurring, confocal systems and two-photon excitation, all exhibit monochromatic and chromatic aberrations. A simple Gaussian model of the aberrated point spread function and optical field for each instrument illustrates spatial distortions and blurring and some unique attenuation effects in confocal and two-photon microscopy. These properties depend on the manner in which illumination and detection combine to give the overall microscope performance and highlight the importance of both optics and sample aberrations; the specimen must be considered as an optical component of the integrated imaging system. Axial focus distortion, from refractive index boundaries at the sample, can be accurately modelled by a geometric ray tracing programme, considering weighted components across the entire objective lens working NA. The modal z-focus error, rather than the average of all weighted rays, agrees closely with empirical measurements of axial focus position from test samples. This agreement is particularly close for confocal measurements when NA 4 weighting is used in the model calculations, but the situation is more complex for samples with non-planar refractive boundaries. Calibration of axial attenuation in a botanical sample, arising from the combination of optical sectioning with specimen-induced spatial distortions and blurring, is possible using an in situ fluorescence sea within a permeabilized preparation. Parametric descriptions of attenuation can be obtained through the guard cell complex of Commelina communis leaf epidermis. Improved images of the 3-D morphology of stomatal guard cells are then obtained by digital correction of attenuation and spatial distortion. Calibrations can be routinely used to correct experimental data by integration with a structured image file format.

Original publication

DOI

10.1046/j.1365-2818.1996.113392.x

Type

Journal article

Journal

Journal of Microscopy

Publication Date

28/03/1996

Volume

181

Pages

99 - 116