[Microbeads and nanobeads in microscopy: a tool for spatial, spectral and dynamic settings. Application to molecular...

This article illustrates a methodology which can be used on cellular and tissular specimens prepared with fluorochromes and analyzed by laser scanning confocal microscopy. Fluorescent beads are used to simulate the fluorochromes and they determine their detectability inside the preparations. They can also be used to verify that the microscopes are set properly so that the resulting images can be analyzed and are reliable to evaluate possible co- localizations. Methods of factor analysis are applied to optical section series obtained on the microscope to characterize the fluorochromes that are used to stain different locations of the specimen. Photophysical properties (emission spectra, decay rates) of the fluorochromes are used to obtain the differentiation. Section series are obtained either by spectral selection via a sequence of filters or by successive scans of the same specimen. Concerning the three- dimensional analysis, differentiation of series that are obtained by z displacement leads to the restoration of focal planes and improves the legibility of specimens. These planes which are not at the same depth when the excitation sources are not aligned can the be superimposed to achieve the co-localization.