Recent improvements in the optics and electronics of flow cytometry systems, as well as in staining techniques, permit the assay of such minute cellular constituents as the DNA and protein contents of micro-organisms. To assess the usefulness of this technique, DNA and protein content distributions were determined in Escherichia coli, Lactobacillus brevis, Lactobacillus casei, Chlorella kessleri 8k, Saccharomyces cerevisiae, Candida utilis, Schizosaccharomyces pombe and Euglena gracilis. Investigations of the DNA content distributions of polyploid strains of Saccharornyces cerevisiae indicated that the method can be used to determine ploidy. The rapidity of flow cytometry measurements allows accurate determinations in large populations.

SummaryWe present a novel technique of far-field localization nanoscopy combining spectral precision distance microscopy with widely used fluorochromes like the Green Fluorescent Protein (GFP) derivatives eGFP, EmGFP, Yellow Fluorescent Protein (YFP) and eYFP, synthetic dyes like Alexa 488 and Alexa 568, as well as fluoresceine derivates. Spectral precision distance microscopy allows the surpassing of conventional resolution limits in fluorescence far-field microscopy by precise object localization after the optical isolation of single signals in time. Based on the principles of this technique, our novel nanoscopic method was realized for laser optical precision localization and image reconstruction with highly enhanced optical resolution in intact cells. This allows for spatial assignment of individual fluorescent molecules with nanometre precision. The technique is based on excitation intensity dependent reversible photobleaching of the molecules used combined with fast time sequential imaging under appropriate focusing conditions. A meaningful advantage of the technique is the simple applicability as a universal tool for imaging and investigations to the major part of already available preparations according to standard protocols. Using the above mentioned fluorophores, the positions of single molecules within cellular structures were determined by visible light with an estimated localization precision down to 3 nm; hence distances in the range of Correspondence to: C. Cremer.

The practical use of flow cytometry is shown in several microbial assays. Recent technical improvements in the optics and electronics of flow cytometric systems as well as in staining techniques permit the measurements of minute cellular components such as the cellular DNA and the protein content of bacteria, algae, moulds and yeasts. Single cell ingredients can be measured by this assay according to their specific stainability. The cell DNA was stained by propidium iodide while the cell protein was fluorochromed by fluorescein-iso-thiocyanate. The DNA synthesis of Saccharomyces cerevisiae and Saccharomyces pastorianus runs discontinuously while the protein content increases continuously during the vegetative growth. The different stages of DNA synthesis of yeast cells can be divided into two 'gap' phases, a synthesis and a mitosis period, corresponding to Howard and Pelc's model of DNA synthesis. Living and dead cells can be counted differentially after staining with Erythrosine B. The red fluorescence of the chlorophyll in algae can readily be used to determine the chlorophyll content of these cells.

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A dual laser beam excitation device for flow analysis of biological particles has been developed. The aid of this arrangement is to increase the range of fluorescent agents employed so far in quantitative and qualitative cytochemistry. Combining an argon ion and a helium-cadmium laser two color fluorescence measurements were performed employing propidium iodide as a DNA stain and fluorescamine which stains total protein in fixed cells. Energy transfer processes between the antibiotic and DNA specific dye mithramycin and propidium iodide both being bound to nuclear chromatin were analyzed. Utilization of energy transfer processes is generally discussed as a mean to extract information about the structure and conformation of nuclear chromatin in situ. The application of a crypton ion laser with three lines near 400 nm and a single line at 350 nm having a light output in each range of nearly one Watt gives the opportunity of utilizing DNA fluorochromes which have an excitation maximum in the deep blue region, DNA spectra are shown employing mithramycin, the benzimidazol derivative 33258 (Hoechst) and the indol compound DAPI which has a high DNA specificity combined with a great stability under UV illumination. By separating two focussed laser beams at their intersecting points with the liquid sample stream the trajectory of each flowing cell crosses the beams sequentially, which causes a solitary dual excitation of each cell. The advantages of a solitary excitation device compared with a simultaneous one is discussed.