Thickness estimation of fluorescent sections using a CSLM

A novel method for the measurement of the section thickness of LM-plastic embedded specimen has been developed. This method makes use of the fluorescence that comes from standard routine stainings such as haematoxylin and eosin and periodic acid-Schiff. An x-z profile of the specimen 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 simple and quick: a slide with five sections takes less than 1 min to measure. A theoretical treatment is presented which shows that the FWHM of the axial fluorescent profile is a good estimate of the actual thickness when the sample thickness is greater than 20 (when thickness is expressed in generalized longitudinal optical coordinates). This corresponds to a thickness of about 1μm when using an NA=1.3 oil-immersion objective and 488-nm excitation. The relative error in thickness is then 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 and insensitive to such problems.