Carbocyanine dye diS-C3(3) was repeatedly employed in monitoring the plasma membrane potential of yeast and other living cells. Four methods of measuring and evaluating probe fluorescence signal were used in different studies, based on following fluorescence parameters: fluorescence intensity emitted within a certain spectral interval, F(580)/F(560) fluorescence emission ratio, wavelength of emission spectrum maximum, and the ratio of respective fluorescence intensities corresponding to the diS-C3(3) bound to cytosolic macromolecules and remaining dissolved in the aqueous cell medium (i.e., unbound, or free). Here we show that data corresponding to the three latter spectral assessments of diS-C3(3) accumulation in cells is mutually convertible, which means that their alternative use cannot lead to ambiguities in the interpretation of the results of biological experiments. On the other hand, experiments based on the effortless measurements of fluorescence intensities should be interpreted cautiously because controversial results can be obtained, depending on the particular choice of cell-to-dye concentration ratio and emission wavelength.

• Keywords
• Fluorescent probe, Plasma membrane potential, Saccharomyces cerevisiae, Spectral analysis, Yeast,
• MeSH
• Fluorescent Dyes chemistry   MeSH
• Spectrometry, Fluorescence methods   MeSH
• Carbocyanines chemistry   MeSH
• Membrane Potentials * MeSH
• Saccharomyces cerevisiae chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Carbocyanines MeSH

The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.

• MeSH
• Fluorescent Dyes chemistry   MeSH
• Carbocyanines chemistry   MeSH
• Membrane Potentials physiology   MeSH
• Rhodotorula cytology   physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Carbocyanines MeSH

Fluorescence emission spectra of yeast cell suspensions stained with calcofluor have recently been identified as promising markers of variations in the quality of yeast cell wall. It is shown in this paper how the raw fluorescence spectra of calcofluor can be transformed to reliable spectral signatures of cell wall quality, which are independent of actual dye-to-cell concentrations of examined cell suspensions. Moreover, the presented approach makes it possible to assess basis fluorescence spectra that allows for the spectral unmixing of raw fluorescence spectra in terms of respective fluorescence contributions of calcofluor solvated in the suspension medium and bound to yeast cell walls.

• MeSH
• Staining and Labeling MeSH
• Benzenesulfonates metabolism   MeSH
• Cell Wall chemistry   metabolism   MeSH
• Fluorescent Dyes metabolism   MeSH
• Spectrometry, Fluorescence MeSH
• Glucose pharmacology   MeSH
• Saccharomyces cerevisiae chemistry   cytology   drug effects   metabolism   MeSH
• Suspensions MeSH
• Dose-Response Relationship, Drug MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Benzenesulfonates MeSH
• C.I. Fluorescent Brightening Agent 28 MeSH Browser
• Fluorescent Dyes MeSH
• Glucose MeSH
• Suspensions MeSH

This review summarizes the main results obtained in the fields of general and molecular microbiology and microbial genetics at the Institute of Microbiology of the Academy of Sciences of the Czech Republic (AS CR) [formerly Czechoslovak Academy of Sciences (CAS)] over more than 50 years. Contribution of the founder of the Institute, academician Ivan Málek, to the introduction of these topics into the scientific program of the Institute of Microbiology and to further development of these studies is also included.

• MeSH
• Academies and Institutes history   MeSH
• History, 20th Century MeSH
• Genetics, Microbial history   MeSH
• Molecular Biology history   MeSH
• Check Tag
• History, 20th Century MeSH
• Publication type
• Journal Article MeSH
• Historical Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Geographicals
• Czech Republic MeSH

Evaluation of emission spectra of fluorescent probes used for the monitoring of membrane potential in microbial cells can be greatly facilitated by using synchronously excited spectroscopy (SES). This method permits the suppression of undesirable spectrum components (contributions due to scattered light or cell autofluorescence) and leads to considerable increase in monitored emission intensity and to narrowing of spectral peaks. It allows an efficient fractional decomposition of the probe fluorescence spectra into their free and bound dye fluorescence components. The usefulness of the method was tested by monitoring the accumulation of the fluorescent membrane potential probe diS-C3(3) in yeast cells, which serves as a qualitative measure of the membrane potential.

• MeSH
• Fluorescent Dyes metabolism   MeSH
• Spectrometry, Fluorescence methods   MeSH
• Carbocyanines metabolism   MeSH
• Membrane Potentials physiology   MeSH
• Saccharomyces cerevisiae physiology   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Carbocyanines MeSH

Three fluorescent probes, tetramethyl rhodamine ethyl ester (TMRE), 3,3'-dipropylthiacarbocyanine iodide (diS-C3(3)) and 3,3'-dipropyloxacarbocyanine iodide (diO-C3(3)), were tested for their suitability as fluorescent indicators of membrane potential in Saccharomyces cerevisiae in studies performed by flow cytometry. For all these dyes the intensity of fluorescence of stained cells increased with probe concentration in the range of 60-3000 nmol/L. The optimum staining period was 15-20 min for diS-C3(3). Depolarization of cells by increased extracellular potassium level and by valinomycin elicited with all probes a drop in fluorescence intensity. In some yeast batches this depolarization was accompanied by a separation of subpopulations with different fluorescence properties.

• MeSH
• Artifacts MeSH
• Benzothiazoles MeSH
• Time Factors MeSH
• Fluorescence MeSH
• Fluorescent Dyes * MeSH
• Fluorometry * MeSH
• Indicators and Reagents MeSH
• Carbocyanines * MeSH
• Membrane Potentials * MeSH
• Organometallic Compounds MeSH
• Flow Cytometry methods   MeSH
• Pyridinium Compounds analysis   metabolism   MeSH
• Scattering, Radiation MeSH
• Saccharomyces cerevisiae physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine MeSH Browser
• 3,3'-dipropyl-2,2'-thiadicarbocyanine MeSH Browser
• 3,3'-dipropyloxadicarbocyanine MeSH Browser
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Benzothiazoles MeSH
• Fluorescent Dyes * MeSH
• Indicators and Reagents MeSH
• Carbocyanines * MeSH
• Organometallic Compounds MeSH
• Pyridinium Compounds MeSH
• tetramethyl rhodamine ethyl ester MeSH Browser