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Selective loading of carboxy SNARF®-1 into mitochondria. BHK cells were loaded with 10 µM carboxy SNARF®-1, AM, acetate (Cat. no. C1271, C1272) for 10 minutes, followed by incubation for 4 hours at room temperature. A) Confocal image (488 nm excitation) of mitochondrial-selective loading of carboxy SNARF®-1 visualized through a 560–600 nm bandpass filter. B) Confocal image of the same cells as in A, but using a 605 nm dichroic mirror and a 610 nm longpass filter. C) Ratio image (A and B) of mitochondria in cells pseudocolored to represent different pH levels. D) Change in mitochondrial pH following the addition of 10 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), resulting in a decrease (acidification) of mitochondrial pH. Image contributed by Brian Herman, University of Texas Health Science Center, San Antonio, and reprinted with permission from Biotechniques 30, 804 (2001).