Due to its spectral characteristics, the fluorochrome nonyl acridine orange (NAO) (&,,:489 nm, X,,:525 nm), which is spontaneously incorporated by mitochondria with a high relative specificity, provides a new probe for the in situ study of these organelles by flow cytometry. In 15 min at 20°C, the dye at 4.75 x M saturates the mitochondrial binding sites present in 1.5 x lo6 cells. Unlike Rh 123, the fixation of the probe is not affected by the action of uncouplers and ionophores. Unlike acridine orange, its binding is not sensitive to nucleases.By studying the mitochondrial incorporation of the fluorochrome during the cell cycle of murine splenocytes, it was possible to show that the biogenesis of NAO-stained mitochondrial constituents mainly occurs during the GI phase.Key terms: Acridines, mitochondria analysis, fluorescent probes, cell cycle, flow cytometry For the past 10 years, the in situ study of mitochondria has been carried out mainly through the use of a mitochondria-specific fluorochrome, rhodamine 123 (Rh 123). Initially, Johnson et al. (17)showed by microscopy that this dye directly and specifically stains the mitochondrial membranes and that its incorporation depends on the mitochondrial transmembrane potential (18).Coupled with analysis by flow cytometry (FCM), probe incorporation has made possible quantitative studies of the mitochondrial compartment (5). Applications were extended to the study of mitochondria in normal and transformed cells (16,21) during proliferation (2,9,10, 15,23,24,26) or differentiation (6,301. Furthermore, rhodamine 123 was used to monitor the effects of various drugs on the transmembrane potential (1,3,14,19). Although these investigations have improved our understanding of the interactions between the nuclear and mitochondrial cell compartments, in particular during division, it was found that fluorochrome incorporation may indicate both a change in the activity of the organelle and a change in its true membrane mass. In order to carry out in situ studies, it was therefore necessary to obtain fluorochromes that bind to mitochondria according to a mechanism independent of respiratory activity.Such a dye would make it possible to determine more accurately than with rhodamine 123 the total mitochondrial mass or the mass of one or several of its constituents, and, in some cases, the size and number of organelles.Among the numerous mitochondria-specific fluorochromes described recently (22,25), it has been shown that 10-nonyl acridine orange (NAO), synthesized by the team of Professor H.W. Zimmermann (12,13,28,29), binds to these organelles in a manner independent of the respiratory activity. Moreover, the spectral properties of NAO allow an optimum excitation at approximately 488 nm, and the fluorescence may be detected between 500 and 600 nm with a n emission maximum at 525 nm.In this study we therefore chose to determine the conditions for use of this fluorochrome in quantitative FCM analysis. NAO is rapidly and spontaneously incorporated by murine splenocytes following c...

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The aim of this study was to investigate the effects on the cell membranes of Escherichia coli of 2.45-GHz microwave (MW) treatment under various conditions with an average temperature of the cell suspension maintained at 37°C in order to examine the possible thermal versus nonthermal effects of short-duration MW exposure. To this purpose, microwave irradiation of bacteria was performed under carefully defined and controlled parameters, resulting in a discontinuous MW exposure in order to maintain the average temperature of the bacterial cell suspensions at 37°C. Escherichia coli cells were exposed to 200-to 2,000-W discontinuous microwave (DW) treatments for different periods of time. For each experiment, conventional heating (CH) in a water bath at 37°C was performed as a control. The effects of DW exposure on cell membranes was investigated using flow cytometry (FCM), after propidium iodide (PI) staining of cells, in addition to the assessment of intracellular protein release in bacterial suspensions. No effect was detected when bacteria were exposed to conventional heating or 200 W, whereas cell membrane integrity was slightly altered when cell suspensions were subjected to powers ranging from 400 to 2,000 W. Thermal characterization suggested that the temperature reached by the microwave-exposed samples for the contact time studied was not high enough to explain the measured modifications of cell membrane integrity. Because the results indicated that the cell response is power dependent, the hypothesis of a specific electromagnetic threshold effect, probably related to the temperature increase, can be advanced.T he interaction of electromagnetic fields (EMFs) and various life processes has been studied and debated for more than half a century. Identifying and evaluating the biological effects of microwaves (MW) is complex and controversial. Whereas one of the current theories is that heat generation induced by microwaves is responsible for biological effects, there has been a persistent view in the physical and engineering sciences that microwave fields are unable to induce bioeffects other than by heating (1) (2). Because of the scarcity of information on the mechanism of interaction between microwave and biological systems, this controversy endures.A great number of studies of the thermal versus nonthermal bioeffects of low-power MW were performed with various cellular functions, including gene expression (3) and mutation (4), enzyme activity (5), unfolding of proteins (6), biochemical cell systems (7), cell wall (8), cell morphology (9), and cell proliferation (10-13). Whereas several authors showed nonthermal effects, safety standards have been set based solely upon the thermal effect of MW. The main reason was that no satisfactory mechanism was proposed to explain the nonthermal bioeffects.When applied at high power, MW bioeffects induced by heating constitute one of the modern approaches for sterilization and decontamination processes in the food industry. In fact, microwaves have long been known to i...