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Journal of Biomedical Optics • Open Access

Multispectral fluorescence imaging to assess pH in biological specimens
Author(s): Matthew R. Hight; Donald D. Nolting; Eliot T. McKinley; Adam D. Lander; Shelby K. Wyatt; Mark Gonyea; Ping Zhao; H. Charles Manning

Paper Abstract

Simple, quantitative assays to measure pH in tissue could improve the study of complicated biological processes and diseases such as cancer. We evaluated multispectral fluorescence imaging (MSFI) to quantify extracellular pH (pHe) in dye-perfused, surgically-resected tumor specimens with commercially available instrumentation. Utilizing a water-soluble organic dye with pH-dependent fluorescence emission (SNARF-4F), we used standard fluorimetry to quantitatively assess the emission properties of the dye as a function of pH. By conducting these studies within the spectroscopic constraints imposed by the appropriate imaging filter set supplied with the imaging system, we determined that correction of the fluorescence emission of deprotonated dye was necessary for accurate determination of pH due to suboptimal excitation. Subsequently, employing a fluorimetry-derived correction factor (CF), MSFI data sets of aqueous dye solutions and tissuelike phantoms could be spectrally unmixed to accurately quantify equilibrium concentrations of protonated (HA) and deprotonated (A;) dye and thus determine solution pH. Finally, we explored the feasibility of MSFI for high-resolution pHe mapping of human colorectal cancer cell-line xenografts. Data presented suggest that MSFI is suitable for quantitative determination of pHe in ex vivo dye-perfused tissue, potentially enabling measurement of pH across a variety of preclinical models of disease.

Paper Details

Date Published: 1 January 2011
PDF: 7 pages
J. Biomed. Opt. 16(1) 016007 doi: 10.1117/1.3533264
Published in: Journal of Biomedical Optics Volume 16, Issue 1
Show Author Affiliations
Matthew R. Hight, Vanderbilt Univ. (United States)
Donald D. Nolting, Vanderbilt Univ. Medical Ctr. (United States)
Eliot T. McKinley, Vanderbilt Univ. Medical Ctr. (United States)
Adam D. Lander, Vanderbilt Univ. (United States)
Shelby K. Wyatt, Vanderbilt Univ. Medical Ctr. (United States)
Mark Gonyea, Vanderbilt Univ. (United States)
Ping Zhao, Vanderbilt Univ. (United States)
H. Charles Manning, Vanderbilt Univ. (United States)


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