Alternative correlative microscopy in the studies of cell nucleus subcompartments

Abstract

A new method combining atomic force (AFM) and confocal laser scanning microscopy (CLSM) and permitting not only to obtain information about the surface topography of nanostructures in the cell nucleus, but to simultaneously identify their molecular composition by using fluorescent dyes and treatment with specific enzymes, is proposed. The content of the vegetative nuclei of the ciliate Paramecium caudatum stained with DAPI for revealing DNA and fluorescently labeled phalloidin specifically binding fibrillar actin was been sequentially studied using CLSM and AFM and by successive analysis with AFM after RNAse and Proteinase K treatments. The proposed method allows one to characterize the surface topography of chromatin and RNP-complexes and reveal nanostructures containing nuclear actin which cannot be visualized using CLSM only.