THICKNESS ESTIMATION OF FLUORESCENT SECTIONS USING A CSLM

A novel method for the measurement of the section thickness of LM-plas tic embedded specimen has been developed. This method makes use of the fluorescence that comes from standard routine stainings such as haema toxylin and eosin and periodic acid-Schiff. An x-z profile of the spec imen is first scanned using a confocal scanning laser microscope, The full-width half-maximum. FWHM, of the profile is computed and used as a measure of the specimen thickness, This method has proven to be simp le and quick: a slide with Eve sections takes less than I min to measu re, A theoretical treatment is presented which shows that the FWHM of the axial fluorescent profile is a good estimate of the actual thickne ss when the sample thickness is greater than 20 (when thickness is exp ressed in generalized longitudinal optical coordinates). This correspo nds to a thickness of about 1 mu m when using an NA=1.3 oil-immersion objective and 488-nm excitation, The relative error in thickness is th en less than 10%. Simulations and experiments have been carried out to examine how problems such as bleaching, sample tilt and curvature of field influence the FWHM. The results show that the method is robust a nd insensitive to such problems.