A new optical multichannel microspectrofluorometer.

A new microspectrofluorometer has been developed that combines a photometric fluorescence microscope with an optical multichannel analyzer. This instrument provides fluorescence emission spectra of biological materials by detecting the entire spectrum simultaneously in real time. These spectra are subsequently recorded and corrected so as to identify the fluorescent reaction products or to test whether fluorescent cytochemical probes bind to the expected substrate within cells. The procedures and advantages of optical multichannel analysis are described, and an application of microspectrofluorometry to acriflavine-Feulgen cytochemistry is given.

7-Amino-actinomycin D as a cytochemical probe. I. Spectral properties.

The optical absorption and fluorescence characteristics of 7-animo-actinomycin D were determined to evaluate its potential as a fluorescent cytochemical probe. At pH 7.0, the absorption maximum and fluorescence excitation maximum are both at 503 nm; the fluorescence emission is at 675 nm. When this compound forms complexes with DNA in solution, the absorption and fluorescence excitation maxima shift to 543 nm and the fluorescence emission shifts to 655 nm. The fluorescence quantum yield is 0.016 for 7-amino-actinomycin D free in solution and 0.01-0.02 for complexes with native DNA. The 7-amino-actinomycin D also exhibits fluorescence shifts characteristic of binding when put into solution with poly(dG-dC) poly(dG-dC), but not with poly(dI-dC) poly(dI-dC). The spectral characteristics are the same at pH 7.0 whether the solvent is 0.01 M PO4 with 0.0001 M EDTA or Earle's salts with 0.025 M N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid.

Evaluation of diallate and triallate herbicides for genotoxic effects in a battery of in vitro and short-term in vivo tests.

Commercial-grade preparations of two thiocarbamate herbicides, diallate and triallate, were evaluated for their mutagenic potential in a battery of short-term bioassays. All in vitro bioassays were performed with and without mammalian metabolic activation, and all such tests were repeated after an interval of at least 1 week. Diallate and triallate were tested in the Salmonella/microsome assay over dose ranges of 0.59 to 118.0 micrograms/plate and 6.37 to 1273 micrograms/plate, respectively. Both diallate and triallate gave positive results in S. typhimurium strains TA1535, TA98, and TA100 only in the presence of a rat-liver metabolic activation system. In Saccharomyces cerevisiae strain D7, diallate was tested at concentrations from 1.18 to 29.50 micrograms/ml, and triallate was tested at 0.955 to 9.548 micrograms/ml. Both diallate and triallate gave negative results for mitotic gene conversion, mitotic crossing-over, and reverse mutation. In the mouse lymphoma L5178Y TK+/- assay, diallate was tested at concentrations ranging from 1 to 72 micrograms/ml, and triallate was tested at 0.5 to 60 micrograms/ml. Both herbicides produced mutagenic responses in the mouse lymphoma assay in the presence of metabolic activation. In the Drosophila sex-linked recessive lethal test, flies were exposed to 0.0004% diallate and 0.001% triallate. In this assay, diallate was considered mutagenic, whereas triallate did not produce a detectable mutagenic response.

Further observations on the chemistry of pararosaniline-Feulgen staining.

Pararosaniline-Feulgen staining of cells in suspension produces nucleus- and chromatin-specific fluorescence as well as color. Experiments were designed to test postulated reaction mechanisms responsible for the fluorescent staining with the nonfluorescent pararosaniline. The reduction in fluorescent-staining intensity by pretreatment of cells with 2.2 x 10-2M K2S2O5 tends to rule out the alkysulfonic acid pathway; conditions favoring the formation of this intermediate reduce staining intensity. The fluorescence enhancement, observed when cells stained in pararosaniline without K2S2O5 are post-treated with K2S2O5, suggests that there is an initial Schiff-base linkage between pararosaniline and an aldehyde of hydrolyzed DNA, and that this linkage is stabilized in the presence of K2S2O5. Microspectrofluorometer measurements of cells stained at various pararosaniline concentrations in 2.2x10-2M K2S2O5, show that the fluorescence emission maximum ranges from about 627 nm at 3.1x10-3 M pararosaniline to about 604 nm at 3.1x10-5M. All of the employed staining protocols appear to produce the same fluorescent product, perhaps a heterocyclic pyronin analog formed from pararosaniline. Flow microfluorometric analysis of cells stained in suspension verified that the relative fluorescence intensity represents relative DNA content. Staining at reduced pararosaniline concentration (3.1x10-4M) reduces the coefficient of variation of the flow microfluorometric histograms, showing that maximum quantitation does not necessarily correlate with maximum staining intensity.