DNA fluorescence induced by polymethine cation pyrvinium binding.

Pyrvinium is a polymethine cation which shows interesting fluorescence emission and DNA binding properties. In diluted aqueous solution, pyrvinium pamoate induced a bright yellow fluorescence in kinetoplast DNA from Trypanosoma cruzi epimastigotes as well as in chicken erythrocyte nuclei under a wide range of excitations. No fading was observed after mounting in suitable media. Spectroscopic studies on pyrvinium solutions revealed bathochromic and hypochromic shifts in the absorption spectrum of its complex with DNA. A striking enhancement of pyrvinium fluorescence was found in solvents of high viscosity or after binding to DNA. Experimental results and the chemical structure of pyrvinium allow us to suggest that the minor groove of adenine-thymine DNA regions could be the specific binding site for this new DNA fluorochrome.

Alkaline hydrolysis/methylation-acetylation: a new technique for ultrastructural DNA cytochemistry.

A new technique for the visualization of DNA-containing structures in electron microscopy is described. Samples of glutaraldehyde-fixed bone marrow from rats were subjected to alkaline hydrolysis to remove RNA and the phosphate of phosphoproteins, followed by a combined blockage of protein carboxyl and amino groups through methylation-acetylation. After uranyl acetate staining of epoxy-embedded ultrathin sections, chromatin from all cell types showed a highly selective and intense electron opacity. Staining methods for DNA were also positive in semithin sections. This simple procedure could be very useful in ultrastructural cytochemistry of DNA and chromatin.

Indium (III)-hematoxylin as a staining and contrasting agent for light and electron microscopy.

A deeply violet indium (III)-hematoxylin complex is formed when indium trichloride is added to an aqueous solution of oxidized hematoxylin. Treatment of glutaraldehyde fixed and Araldite embedded sections of rat seminiferous tubules with indium-hematoxylin revealed a definite staining and contrasting pattern. Semithin sections showed chromatin and nucleoli in violet-blue. Under the electron microscope, chromatin, nucleoli, ribosomes, synaptonemal complexes, chromatoid bodies, membranous components, and microtubules from sperm tails presented high electron opacity, while the acrosome and basement membrane appeared with a lower contrast. This performed indium-hematoxylin complex, which shows an absorption peak at lambda = 560 nm with shoulders at about lambda = 440 and 400 nm, could be valuable as a new staining and electron contrasting agent.

New fluorescence reactions in DNA cytochemistry. 1. Microscopic and spectroscopic studies on nonrigid fluorochromes.

Some nonrigid DNA-binding antibiotics and fluorochromes that recognize adenine-thymine (AT) sequences are widely applied in biomedical research, but the microscopic use, spectral characteristics and DNA binding modes of other similar compounds have been overlooked or scarcely explored. After treatment with thioflavine T, auramine O and G, curcumin, bis-aminophenyl-oxadiazol, berenil and distamycin A, a bright DNA-dependent fluorescence reaction was found in the chromatin of interphase nuclei, meiotic and polytene chromosomes, spermatozoa heads and kinetoplasts of Trypanosoma cruzi epimastigotes. Nucleoli and basophilic cytoplasm showed low or no fluorescence; the highest emission occurred in the AT-rich kinetoplast DNA. When bound to DNA or in the presence of alpha-cyclodextrin and viscous solvents or cosolutes, nonrigid compounds revealed a striking enhancement of fluorescence. The results indicate that these new or poorly known fluorochromes bind selectively to DNA-containing structures and that the minor groove from AT-rich DNA regions could represent the specific and highly fluorescent binding site.

New fluorescence reactions in DNA cytochemistry. 2. Microscopic and spectroscopic studies on fluorescent aluminum complexes.

Metal-dye complexes are widely applied in light microscopic techniques for chromatin staining (e.g., hematoxylin and carmine), but fluorescent complexes between phosphate-binding cations and suitable ligands have been little used. Preformed and postformed Al complexes with different anionic dyes induced strong and selective fluorescence reactions in nuclei from chicken blood smears, frozen sections, paraffin-embedded sections and Epon-embedded sections of mouse and rat tissues, mitotic chromosomes, meiotic chromosomes and kinetoplasts of Trypanosoma cruzi epimastigotes. The DNA-dependent fluorescence of these structures showed a very low fading rate. The emission colors were related to the ligand. The most suitable compounds for forming fluorescent Al chelates were 8-hydroxyquinoline, morin, nuclear fast red and purpurin. Staining with diluted carmine solutions and InCl3 mordanting, followed by 8-hydroxyquinoline, also induced chromatin fluorescence. After treating isolated mouse chromosomes with the preformed complex Al-nuclear fast red, x-ray microanalysis indicated a P:Al:dye binding ratio of about 40:15:1. The selectivity, stability and easy formation of these fluorescent Al complexes are obvious advantages for their use as new cytochemical probes in cytologic studies.

Chromatin fluorescence after carmine staining.

After staining with dilute solutions (0.1 mg/ml in distilled water) of commercial carmine, a strong reddish orange fluorescence was observed in nuclei from cell smears and frozen and paraffin tissue sections. Optimal exciting light was 436 nm (violet-blue) or 450-490 nm (blue). Compact chromatin from interphase nuclei, mitotic and meiotic chromosomes and the kinetoplast of Trypanosoma cruzi showed the highest fluorescence, while the basophilic cytoplasm appeared weakly fluorescent. No emission was observed in cartilage matrix, mast cell granules or goblet cell mucin. This selective method could be valuable in microscopic and cytochemical studies on chromatin because the carmine fluorescence is stable and preparations can be dehydrated and mounted permanently without changes in the fluorescence pattern.

Aluminium binding to chromatin DNA as revealed by formation of fluorescent complexes with 8-hydroxyquinoline and other ligands.

After aluminium mordanting of chicken blood smears, several chelating dyes and reagents were tested in order to detect the metal binding to chromatin DNA by fluorescence microscopy. 8-Hydroxyquinoline induced the most selective fluorescence reaction, which was abolished after DNase or trichloroacetic acid treatment. Morin, purpurin and nuclear fast red showed an intense nuclear fluorescence, but lower selectivity. Fluorescence was also observed in chromatin treated with morin or curcumin without mordanting. The simplicity and microscopical characteristics of the A1/8-hydroxyquinoline fluorescence reaction make it very suitable for cytological studies of chromatin.

Fluorescence reaction of chromatin by curcumin.

Treatment of cell smears, paraffin, and Epon tissue sections with aqueous solutions of curcumin results in a green fluorescence reaction in chromatin under violet-blue excitation which is abolished after extraction procedures with DNase and TCA. The selective fluorescence characteristics of curcumin support the possibility of employing this dye as a new fluorochrome.

Influence of fixation, exciting light and section thickness on the primary fluorescence of samples for microfluorometric analysis.

The primary fluorescence (autofluorescence) of some cell and tissue components depends on the fixative and fixing time, as well as on the thickness of paraffin sections and the wavelength of exciting light. The highest autofluorescence emission (pale green) was observed by using violet-blue excitation. After aldehyde fixation, the autofluorescence of some tissue structures was higher than after methanol or ethanolacetic acid. These features must be taken into account when fluorescence microscopy is applied to the study of cell smears and paraffin embedded tissues after flurochroming or immunofluorescence reactions.