. "10.1371/journal.pone.0033229" . . "7" . "In Vivo Determination of Organellar pH Using a Universal Wavelength-Based Confocal Microscopy Approach"@en . . "In Vivo Determination of Organellar pH Using a Universal Wavelength-Based Confocal Microscopy Approach"@en . "3"^^ . . "3"^^ . "In Vivo Determination of Organellar pH Using a Universal Wavelength-Based Confocal Microscopy Approach" . "V\u00E1chov\u00E1, Libu\u0161e" . "12"^^ . "In Vivo Determination of Organellar pH Using a Universal Wavelength-Based Confocal Microscopy Approach" . "Pineda Rod\u00F3, Albert" . "RIV/00216208:11310/12:10118891!RIV13-GA0-11310___" . . . . "[216138C4FCF9]" . . "subcellular metabolic functions; pH-dependent ratiometric fluorescent proteins; GFP; organellar pH; confocal microscopy"@en . "http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0033229" . . "3" . . "Many essential cellular processes are affected by transmembrane H+ gradients and intracellular pH (pHi). The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study."@en . "11310" . . "1932-6203" . "Many essential cellular processes are affected by transmembrane H+ gradients and intracellular pH (pHi). The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study." . . . . "141052" . "PLoS ONE" . . . "RIV/00216208:11310/12:10118891" . . "000303857100023" . "Palkov\u00E1, Zdena" . . "I, P(GA204/08/0718), P(LC06063), Z(AV0Z50200510), Z(MSM0021620858)" . "US - Spojen\u00E9 st\u00E1ty americk\u00E9" . . "Pineda Rod\u00F3, Albert" . . . . . .