Fluorescence Microscopy and Fluorophores |
Acridin orange (AO) is a very simple selective, not a specific simultaneous stain for DNA and RNA. The staining pattern of AO (green fluorescence for DNA, red fluorescence for RNA) is a function of the dye concentration, which has to be adjusted in such a way that AO bound to DNA is a monomeric intercalation form, whereas AO bound to RNA is a complex of dye polymers and the RNA. AO can be used as a vital stain without fixation of the cells. Depending on the buffer used for the dilution of the dye and the pH AO can also stain lysosomes. The absorption of AO is in between 440 and 480 nm (blue), the emission is in between 520 nm (green for DNA) and 650 nm (orange for RNA).
References: Shapiro, H.M., Practical Flow Cytometry, A.R. Liss Inc., New York 1988; Romeis, B., Mikroskopische Technik, Oldenbourg Verlag, Munich 1968 (in German); Stockert, J.C., and J.A. Lisanti, Chromosoma 37, 117-130 (1972)
DiOC (correct DiOC6(3) i.e., 3,3'-dihexyloxacarbocyanine iodide) is a lipophilic carobocyanine dye preferentially staining certain membrane structures in living cells. It can be used to demonstrate the dynamics of the endoplasmic reticulum. It works with living cells as well as in cells slightly fixed in formaldehyde or glutaraldehyde. Other fixatives containg methanol, ethanol or acetone dissolve the lipids in the membrane. In these cases you will not get a fluorescence from membranes with DiOC. DiOC has an absorption maximum around 483 nm (blue) and an emission at about 600 nm.
References: Terasaki et al., Cell 38, 101-108 (1984); Lee and Chen, Cell 54, 37-46 (1988); Dabora and Sheetz, Cell 54, 27-35 (1988)
Rhodamine 123 has been termed a mitochondria-specific dye, because this cationic cyanine dye is accumulated in electrically negative compartments such as mitochondria in healthy cells. Uncoupling agents and inhibitors reduce mitochondrial fluorescence. The large membrane surface area in the mitochondrial matrix may contribute to the staining by binding large amounts of accumulated probe. Rhodamine 123 has an absorption maximum at about 485 nm (blue) and an emission maximum at 530 nm.
References: Johnson et al., J. Cell Biol. 88, 526-535 (1981); Waggoner, A.S., in: Applications of Fluorescence in the Biomedical Sciences (L.Taylor, A.Waggoner, R.Murphy, F.Lanni, R.Birge, eds.), pp-3-28, Alan Liss, 1986.
The hydrophobic dye Nile Red has been shown to be highly selective for lipid vesicles in cells. Depending upon the relative hydrophobicity of the solvent used, the excitation and emission maxima of nile red fluorescence can vary over a range of more than 60 nm (excitation 490-550 nm, emission 530-610 nm)
References: Greenspan et al., J. Cell Biol. 100, 965-973 (1985); Waggoner, A.S., in: Applications of Fluorescence in the Biomedical Sciences (L. Taylor, A. Waggoner, R. Murphy, F.Lanni, R. Birge, eds.) pp. 3-28, Alan Liss, 1986.
DAPI (4',6-Diamidino-2-phenylindole) forms fluorescent complexes with AT-rich sequences of double-stranded DNA and is a widely used brilliant and stable stain for DNA, especially for fixed cells. The absorption maximum is at 344 nm, the emission maximum at 449 nm.
References: Hamada and Fujita, Histochemistry 79, 219-226 (1983); Coleman and Goff, Stain Technol. 60, 145-161 (1985); Otto and Tsou, Stain Technol. 60, 7-11 (1985).
Hoechst 33342 (bisbenzimide H 33342) is a specific stain for AT-rich regions of double-stranded DNA like DAPI and also the fluorescence properties are similar to DAPI. It can be used preferentially with living, unfixed cells. The absorption maximum is at 340 nm, the emission maximum at 450 nm.
References: H.Shapiro, Cytometry 2, 143-150 (1981); G.Holmquist, Chromosoma 49, 333-356 (1975).
The combination of Pyronin Y as a general fluorescent dye for nucleic acids (DNA and RNA) with DAPI or Hoechst 33342 opens the possibility to show the fluorescence of DNA and RNA in the same specimen. In a first step, the DNA is stained with DAPI or Hoechst 33342 as described above. After some minutes pyronine Y is added to the cells. As the DNA binding regions are covered by DAPI or the Hoechst, pyronine can only react with RNA. The absorption and emission wavelengths of both DAPI or Hoechst and pyronine are well separated, the detection of the two fluorescence signals can occur simultaneously using a double filter set in the microscope.
References: H. Shapiro, Practical Flow Cytometry, A.R.Liss, New York 1988.
As mentioned above, the DNA-specific dyes DAPI or Hoechst can give a faint cytoplasmic background staining if used in higher concentrations needed for a strong fluorescence. For a prevention of the background fluorescence one can block the cytoplasmic proteins with sulforhodamine 101 and stains the cells afterwards with DAPI. Sulforhodamine 101 has an absorption maximum at 490 nm and an emission maximum at 615 nm. Nevertheless, using the UV line for DAPI fluorescence (369 nm), sulforhodamine gives a characteristic red fluorescence pattern of the cytoplasm which contrasts nicely to the blue fluorescence of the cell nucleus.
One of the most exciting efforts in the field of cell biology is the labelling of proteins with GFP-molecules and their mutated variants. This method allows the detection of single protein molecules in the living cells as well as in fixed samples. Since the discovery and cloning of the wild-type GFP from the jellyfish Aequorea victoria several chromophores (fluorophore) variants have been detected. And, moreover, a new red fluorescent protein from Discosoma spec. has been discoverd. The most important variants of fluorescent proteins are listed below:
These tools - especially in the different combinations - offer an excellent possibility to study and to monitor dynamic processes as well as different interactions between cellular components in living cells. The strategy of this technique is as follows: after plasmid construction (protein of interest or a suitable fragment of it together with the desired fluorescent protein gene) appropriate cells can be transfected. The light stimulated fluorescence of these molecules is independent of cofactors, further substrates or other gene products. It is possible to establish permanent cell lines expressing one or more tagged proteins of interest, which is obviously the great advantage of this method.