Imaging of Mitochondrial pH Using SNARF-1
Year: 2011
Type: book-chapter
Abstract: Laser scanning confocal microscopy provides the ability to image submicron sections in living cells and tissues. In conjunction with pH-indicating fluorescent probes, confocal microscopy can be used to visualize the distribution of pH inside living cells. Here, we describe a confocal microscopic technique to image intracellular pH in living cells using SNARF-1, a ratiometric pH-indicating fluorescent probe. SNARF-1 is ester-loaded into the cytosol and mitochondria of adult cardiac myocytes. Using 568-nm excitation, emitted fluorescence longer and shorter than 595-nm are imaged and then ratioed after background subtraction. Ratio values for each pixel are converted to values of pH using a standard curve (lookup table). Images of the intracellular distribution of pH show cytosolic and nuclear areas to have a pH of ∼7.1, but in regions corresponding to mitochondria, pH is 8.0, giving a mitochondrial ΔpH of 0.9. During hypoxia, mitochondrial pH decreases to cytosolic values, signifying the collapse of ΔpH. These results illustrate the ability of laser scanning confocal microscopy to image the intracellular distribution of pH in living cells and to determine mitochondrial ΔpH.
Source: Methods in molecular biology
Authors: Venkat K. Ramshesh, John J. Lemasters
Institution: Medical University of South Carolina
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: green
Awards: G1081131570, G1229030733, G1436149861, G2108293184, G5010878381, Mitochondrial Dysfunction and Drug Hepatotoxicity, South Carolina COBRE in Oxidants, Redox Balance and Stress Signaling, G8013564434, Ceramide Signaling in the Regulation of Head & Neck Cancer Cell Death and Therapy, Medical University of South Carolina - Cancer Center Support Grant
OpenAlex ID: W355036