Microscopic investigation of cell cultures is one of the most commonly used methods in cell biology. It is used to evaluate cell morphology, growth and structural changes. For such examinations, suitable staining methods are often investigated. Several dyes, however, have proven problematic for use on cells grown on nanofibrous or 3D-printed biomaterials because they stain the substrate in a similar way to the cells. This suggests optimization of the staining protocol.

An aspect often neglected in such microscopic evaluations is the choice of filters and similar optical modifications of the microscope. The microscopic setup, however, can significantly affect the image quality for diverse fluorescent and other dyes.

Here we provide an overview of the influence of diverse microscopic setups, using 3T3-Swiss albino mouse embryonic fibroblasts stained with hematoxylin and eosin (H&E), one of the most commonly used standard histological staining techniques. While hematoxylin stains basophilic structures such as cell nuclei blue-violet, eosin stains acidophilic parts of the cells pink, especially the cytoplasm.

A fluorescence microscope Axio Observer 7 (Zeiss) was used to examine the cells in transmitted light, comparing the usual transmitted light brightfield images with photographs taken using different phase contrast modes, differential interference contrasts (DIC), crossed polarizers, etc. In addition, different exposure times, condenser positions, and white balances were compared.

The results show how important it is to carefully select the microscopic setup in order to optimize the quality of images of stained mammalian cells.