Multispectral fluorescence imaging to assess pH in biological specimens.

Hight MR, Nolting DD, McKinley ET, Lander AD, Wyatt SK, Gonyea M, Zhao P, Manning HC
J Biomed Opt. 2011 16 (1): 016007

PMID: 21280913 · PMCID: PMC3041815 · DOI:10.1117/1.3533264

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.

MeSH Terms (9)

Animals Cell Line, Tumor Colorectal Neoplasms Hydrogen-Ion Concentration Mice Mice, Nude Microscopy, Fluorescence, Multiphoton Reproducibility of Results Sensitivity and Specificity

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