12 September 2003 Analysis of intracellular pH (pHcyt) in mouse models of angiogenesis and carcinogenesis by spectral imaging microscopy, real-time confocal imaging microscopy, and multiphoton spectral imaging
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Abstract
We have shown that a specific cytosolic pH (pHcyt) regulatory mechanism, i.e., vacuolar type H+-ATPases at the plasma membrane (pmV-ATPases), allows angiogenic and metastatic cells to survive in an acidic and hostile environment. However, a functional evaluation of this pump's activity in situ (i.e., in living animal models) has not been attempted. We developed a mouse model of angiogenesis and metastasis based on the dorsal skin fold chamber, and implanted highly metastatic human tumor cells that have been engineered to express green fluorescent protein (GFP). GFP can be used as a pH reporter because its fluorescence is pH sensitive. Our studies in isolated single cells indicated that there are distinct pHcyt gradients in the invadipodia versus the lamellipodia due to the preferential expression of pmV-ATPases at the leading edge. We hypothesize that in vivo, these pH gradients also exist. We employed spectral imaging and real time confocal imaging microscopy, since these approaches are complementary and exhibited unsurpassed temporal and spectral resolution, thus allowing us to study pHcyt in discrete subcellular regions of the cells expressing GFP. We can acquire a full frame (i.e., 512 x 512 pixels) in real time confocal imaging at ca. 25-50 msec, whereas spectral imaging allow us to obtain spectral information from discrete domains of ca. 10 μm in the x-y plane and every 10 μm from leading to lagging edge within a time frame of 5 msec at 0.4 nm spectral resolution. This is possible because we employ frame transfer cooled CCD cameras and spectrographs. Studies are under way to evaluate proton gradients using multiphoton approaches since this will allow us to evaluate pH deeper into the tissue (i.e., 300-600 μm), and should allow us to follow pHcyt and the progression of tumor metastasis.
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Souad Sennoune, Debasish Maiti, Raul Martinez-Zaguilan, "Analysis of intracellular pH (pHcyt) in mouse models of angiogenesis and carcinogenesis by spectral imaging microscopy, real-time confocal imaging microscopy, and multiphoton spectral imaging", Proc. SPIE 4967, Genetically Engineered and Optical Probes for Biomedical Applications, (12 September 2003); doi: 10.1117/12.478401; https://doi.org/10.1117/12.478401
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