MitoTracker dyes are retained quite well after cell fixation in formaldehyde and can often withstand lipophilic permeabilizing agents. In contrast, the MitoFluor probes are designed specifically for actively respiring cells and are not suitable for fixation and counterstaining procedures. Another popular mitochondrial probe, entitled JC-1, is useful as an indicator of membrane potential and in multiple staining experiments with fixed cells. This carbocyanine dye exhibits green fluorescence at low concentrations, but can undergo intramolecular association within active mitochondria to produce a shift in emission to longer (red) wavelengths. The change in emission wavelength is useful in determining the ratio of active to non-active mitochondria in living cells.

In the digital image featured above, the mitochondria present in a culture of COS-7 kidney fibroblasts are easily observable due to staining with MitoTracker Red CMXRos. The filamentous actin and cell nuclei present in the culture, which were respectively labeled with Alexa Fluor 488 conjugated to phalloidin and Hoechst 33258, are also readily apparent. 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.