Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range.

We 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 10-30 nm were resolved between individual fluorescent molecules allowing to apply different quantitative structure analysis tools.

Automated microaxial tomography of cell nuclei after specific labelling by fluorescence in situ hybridisation.

Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem the Heidelberg 2pi-tilting device has been mounted on the Brno high-resolution cytometer as an attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre.

Microbial determinations by flow cytometry.

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 Saccharomyces cerevisiae indicated that the method can be used to determine ploidy. The rapidity of flow cytometry measurements allows accurate determinations in large populations.

Flow cytometric determinations of cellular substances in algae, bacteria, moulds and yeasts.

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.

Extended application of flow microfluorometry by means of dual laser excitation.

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.