Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-spZc stains that are retained in fted, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and ation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding pattems, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellat regions. Therefore, these stains provide cMxR0s-H~

Exposure of mammalian cells to either ionizing radiation or mutagenic and carcinogenic substances can induce chromosome aberrations. These aberrations in turn may give rise to micronuclei which can be found in cells during the interphase after division. A two-step method is presented that allows separation of micronuclei from cell nuclei. They can then be measured and analysed according to their DNA content in a flow cytometer. The method involves an initial detergent treatment of cells followed by a second treatment with sucrose and citric acid. Micronuclei with DNA content larger than 2% of the G1-nuclei can be measured. The method is tested and compared with microscopic observations of micronucleated cells in irradiated, asynchronous, and synchronized Ehrlich ascites tumour cells growing in vitro. The agreement between the flow cytometric technique and microscopic observations is excellent when the dose-dependent number of micronuclei per cell is taken into consideration.

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SummaryWe compared fluorescent signals obtained with fluorescein conjugates and the ELF-97 (enzyme-labelled fluorescence) phosphatase substrate [2-(5′-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone] in labelling cytological structures requiring high spatial resolution. Enzymatic cleavage of the ELF-97 phosphatase substrate yields an extremely fine precipitate that remains well localized to the site of enzymatic activity. This precipitate fluoresces bright yellow-green, with maximal excitation at ~360 nm and maximal emission at 530 nm. The ELF substrate was used with streptavidinalkaline phosphatase, to fluorescently label site-specific probes bound to their targets, including cell-surface sites, cytoplasmic organelles, nuclear antigens and cytoskeletal networks. All targets were labelled successfully with both the ELF substrate and fluoresceinated probes or protein conjugates. However, the ELF method was frequently more sensitive, with lower background fluorescence, allowing detection of more lysosomes, actin filaments, microtubules and nuclear targets than were visible with corresponding fluoresceinated probes. The ELF substrate was also used with antifluorescein-alkaline phosphatase to amplify fluorescein signals. We found that the ELF signal was in all cases brighter and more photostable than fluorescein signals, permitting shorter film exposures and allowing more time for examining samples. Surprisingly, relative brightness and photostability depended on the target, rather than being a general phenomenon related to the choice of dye alone.

DNA synthesis in radiation-induced micronuclei of Chinese hamster cells was studied as a function of time after irradiation using pulse labelling of cells with bromodeoxyuridine (BrdUrd) and an immunofluorescence technique with anti-BrdUrd antibodies. It was shown with this technique that DNA synthesis in micronuclei corresponds with DNA synthesis in nuclei during S phase in approximately 98% of the micronuclei. The presence of radiation-induced micronuclei that are too large to be produced by acentric fragments alone was therefore attributed at least in part to DNA synthesis in micronuclei. A partially synchronized progression of micronuclei from G1 phase into S phase could be observed allowing the measurement of the duration of G1 phase in cells containing micronuclei. The duration of G1 phase in these cells agreed with the duration of G1 phase in unirradiated cells.

The distribution of the DNA content of radiation-induced micronuclei was analysed in several cell lines (Chinese hamster, Syrian hamster and mouse NIH-3T3 cells) by flow cytometry. Frequency and DNA content of micronuclei were measured simultaneously using fluorescence and forward scatter signals of micronuclei and nuclei in suspension stained with ethidium bromide. Computerized random breakage of chromosomes and random combination of fragments was performed to compare the measured micronucleus distributions in synchronized cells irradiated during G1-phase with calculated distributions. The measured DNA distribution of radiation-induced micronuclei was found to be influenced by several factors: (1) the DNA distribution and the centromeric index of the chromosomes in the various cell lines; (2) the cell cycle phase at time of micronucleus measurement due to DNA synthesis in micronuclei; (3) the presence of chromosome fragments in micronuclei; and (4) the presence of whole chromosomes in micronuclei. These factors were shown to be responsible for the previously found large radiation-induced micronuclei which could not be explained by the classic assumption only that radiation-induced micronuclei are mainly produced by single acentric fragments.

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