%0 Journal Article
%T In Vivo Determination of Organellar pH Using a Universal Wavelength-Based Confocal Microscopy Approach
%A Albert Pineda Rod¨®
%A Libu£¿e V¨¢chov¨¢
%A Zdena Palkov¨¢
%J PLOS ONE
%D 2012
%I Public Library of Science (PLoS)
%R 10.1371/journal.pone.0033229
%X 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.
%U http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0033229