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.

• Klíčová slova
• Fluorescent probe, Plasma membrane potential, Saccharomyces cerevisiae, Spectral analysis, Yeast,
• MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční spektrometrie metody   MeSH
• karbocyaniny chemie   MeSH
• membránové potenciály * MeSH
• Saccharomyces cerevisiae chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3,3'-dipropylthiacarbocyanine MeSH Prohlížeč
• fluorescenční barviva MeSH
• karbocyaniny 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
• fluorescenční barviva chemie   MeSH
• karbocyaniny chemie   MeSH
• membránové potenciály fyziologie   MeSH
• Rhodotorula cytologie   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3,3'-dipropylthiacarbocyanine MeSH Prohlížeč
• fluorescenční barviva MeSH
• karbocyaniny 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
• barvení a značení MeSH
• benzensulfonáty metabolismus   MeSH
• buněčná stěna chemie   metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• glukosa farmakologie   MeSH
• Saccharomyces cerevisiae chemie   cytologie   účinky léků   metabolismus   MeSH
• suspenze MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzensulfonáty MeSH
• C.I. Fluorescent Brightening Agent 28 MeSH Prohlížeč
• fluorescenční barviva MeSH
• glukosa MeSH
• suspenze 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
• akademie a ústavy dějiny   MeSH
• dějiny 20. století MeSH
• mikrobiální genetika dějiny   MeSH
• molekulární biologie dějiny   MeSH
• Check Tag
• dějiny 20. století MeSH
• Publikační typ
• časopisecké články MeSH
• historické články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Geografické názvy
• Česká republika 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
• fluorescenční barviva metabolismus   MeSH
• fluorescenční spektrometrie metody   MeSH
• karbocyaniny metabolismus   MeSH
• membránové potenciály fyziologie   MeSH
• Saccharomyces cerevisiae fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3,3'-dipropylthiacarbocyanine MeSH Prohlížeč
• fluorescenční barviva MeSH
• karbocyaniny 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
• artefakty MeSH
• benzothiazoly MeSH
• časové faktory MeSH
• fluorescence MeSH
• fluorescenční barviva * MeSH
• fluorometrie * MeSH
• indikátory a reagencie MeSH
• karbocyaniny * MeSH
• membránové potenciály * MeSH
• organokovové sloučeniny MeSH
• průtoková cytometrie metody   MeSH
• pyridinové sloučeniny analýza   metabolismus   MeSH
• radiační rozptyl MeSH
• Saccharomyces cerevisiae fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine MeSH Prohlížeč
• 3,3'-dipropyl-2,2'-thiadicarbocyanine MeSH Prohlížeč
• 3,3'-dipropyloxadicarbocyanine MeSH Prohlížeč
• 3,3'-dipropylthiacarbocyanine MeSH Prohlížeč
• benzothiazoly MeSH
• fluorescenční barviva * MeSH
• indikátory a reagencie MeSH
• karbocyaniny * MeSH
• organokovové sloučeniny MeSH
• pyridinové sloučeniny MeSH
• tetramethyl rhodamine ethyl ester MeSH Prohlížeč