Attempts to shift the absorption and emission spectra of Aequorea victoria fluorescent proteins to wavelengths in the orange and red regions of the spectrum have met with little success. However, fluorescent proteins from other marine species have enabled investigators to extend the available spectral regions to well within the red wavelength range. The DsRed fluorescent protein and its derivatives, originally isolated from the sea anemone Discosoma striata, are currently the most popular analogs for fluorescence analysis in the 575 to 650-nanometer region. Another protein, HcRed from the Heteractis crispa purple anemone, is also a promising candidate for investigations in the longer wavelengths of the visible spectrum. Newly developed photoactivation fluorescent proteins, including photoactivatable green fluorescent protein (PA-GFP), Kaede, and kindling fluorescent protein 1 (KFP1), exhibit dramatic improvements over GFP (up to several thousand-fold) in fluorescence intensity when stimulated by violet laser illumination. These probes should prove useful in fluorescence confocal studies involving selective irradiation of specific target regions and the subsequent kinetic analysis of diffusional mobility and compartmental residency time of fusion proteins.

Log phase 3T3 cells (illustrated above) were transfected with a recombinant plasmid vector containing a chimeric fusion gene product of DsRed fluorescent protein and the mitochondrial targeting sequence from subunit VIII of human cytochrome C oxidase. Stable transfectants were fixed, permeabilized, and treated with phalloidin conjugated to Alexa Fluor 488 before being counterstained with 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.