Fluorophores targeted at specific intracellular organelles, such as the mitochondria, lysosomes, Golgi apparatus, and endoplasmic reticulum, are useful for monitoring a variety of biological processes in living cells using confocal and fluorescence microscopy. In general, organelle probes consist of a fluorochrome nucleus attached to a target-specific moiety that assists in localizing the fluorophore through covalent, electrostatic, hydrophobic or similar types of bonds. Many of the fluorescent probes designed for selecting organelles are able to permeate or sequester within the cell membrane (and therefore, are useful in living cells), while others must be installed using monoclonal antibodies with traditional immunocytochemistry techniques. In living cells, organelle probes are useful for investigating transport, respiration, mitosis, apoptosis, protein degradation, acidic compartments, and membrane phenomena. Cell impermeant fluorophore applications include nuclear functions, cytoskeletal structure, organelle detection, and probes for membrane integrity. In many cases, living cells that have been labeled with permeant probes can subsequently be fixed and counterstained with additional fluorophores in multicolor labeling experiments.

Using the popular triple fluorophore combination of MitoTracker Red CMXRos, Alexa Fluor 488 conjugated to phalloidin, and Hoechst 33258, the adherent culture of rat kidney mesangial cells illustrated above was stained to visualize the mitochondria, filamentous actin network, and nuclei. 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.