title: Multispectral fluorescence imaging to assess pH in biological specimens.
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.
authors.affiliation: Vanderbilt University, Department of Physics, Nashville, Tennessee 37221, USA.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Hight
authors.forename: Donald D
authors.initials: DD
authors.lastname: Nolting
authors.forename: Eliot T
authors.initials: ET
authors.lastname: McKinley
authors.forename: Adam D
authors.initials: AD
authors.lastname: Lander
authors.forename: Shelby K
authors.initials: SK
authors.lastname: Wyatt
authors.forename: Mark
authors.initials: M
authors.lastname: Gonyea
authors.forename: Ping
authors.initials: P
authors.lastname: Zhao
authors.forename: H Charles
authors.initials: HC
authors.lastname: Manning
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000737:chemistry* / Q000175:diagnosis*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008819:Mice, Nude; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity