An in-house constructed dual channel confocal fluorescence microscope for biomolecular imaging

Abstract

The confocal fluorescence microscope is an essential live cell imaging tool in bioscience research. Several experimental investigations in the field of biomedical research require a dedicated confocal fluorescence microscope. However, commercial confocal microscopes are prohibitively expensive for many individual laboratories and they often have an inflexible design not amenable to user desired modifications. Here we report on the design, development, and calibration of a cost-effective dual channel confocal fluorescence microscope that can capture two biological events simultaneously. The microscope is successfully employed to image and study the simultaneously occurring active and passive transport of molecules across the nuclear membrane. Passive diffusion of FITC labelled dextran molecules are monitored along with the active transport of gold nanoparticles of diameter 20 nm in the time-lapse imaging mode. The experiments carried out in digitonin permeabilized HeLa cells indicate that both active and passive nuclear transport pathways coexist together.