The network of fibrous actin filaments (also commonly referred to as microfilaments) found in the vast majority of mammalian cell lines appears as a complex interconnected organization of linear bundles aligned in two-dimensional arrays or as a three-dimensional matrix. These protein fibers are easily labeled with synthetic or natural fluorophores and can be subsequently visualized using fluorescence microscopy with the appropriate filter combination. Plasmid pEGFP-Actin vector gene product expression in various cell types (from both transiently and stably transfected clones) occurs due to the efficient intracellular translation of a fusion nucleotide sequence combining the enhanced green fluorescent protein domain with a functional copy of human cytoplasmic beta-actin. When expressed in living cells, the gene product is incorporated into actin filaments without significant structural interference from the EGFP protein moiety. The fluorescence excitation maximum of EGFP is 488 nanometers and the corresponding emission maximum occurs at 507 nanometers, with a relatively high (approximately 0.60) fluorescence quantum yield.

The rat thoracic aorta (A-10) cells illustrated above were transfected with a chimeric EGFP plasmid vector that expresses a fluorescent fusion protein targeted at cytoplasmic actin. The fusion protein is incorporated into the growing actin filament network to enable visualization of actin-containing subcellular structures in living and fixed cells. The specimen illustrated in this section was also stained for mitochondria with MitoTracker Red CMXRos, and for DNA in the nucleus with the ultraviolet-absorbing probe DAPI. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.