Cytosolic pH Gradients in Cultured Neuronal Cell Lines Studied by Laser Scanning Confocal Microscopy, Real Time Confocal Microscopy, and Spectral Imaging Microscopy

摘要

Changes in intracellular pH are important for the regulation of many physiological processes including: cell growth and differentiation, exocytosis, synaptic transmission, cell motility and invasion, to name a few. In pathological states such as cancer and diabetes, pH regulation is known to be altered. Nevertheless the physiological and pathological significance of this ion, there are still many gaps in our knowledge. The advent of fluorescent pH probes to monitor this ion, has substantially accelerated its study. New advances in the methods of detection of this ion by fluorescence-based approaches have also helped us to understand more about the regulation of cytosolic pH. This study evaluates the usefulness of real time confocal imaging microscopy, laser scanning confocal microscopy, and spectral imaging microscopy to the study of pH. These approaches exhibit unsurpassed temporal, spatial, and spectral resolution and are complementary. We employed cell lines derived from the brain (glioma and neuroblastoma) exhibiting soma and dendrites. The existence of cell polarity suggests that the different protein composition/microenvironment in discrete subcellular domains may affect the properties of fluorescent ion indicators. We performed in situ calibrations of pH probes in discrete cellular regions of the neuronal cell lines to eliminate any bias in data interpretation because of differences in cell thickness/microenvironment (i.e. viscosity or differential dye/protein interactions). We show that there are distinct in situ calibration parameters in different cellular domains. These indicate that in situ titrations in discrete cellular domains are needed to assign pH values. We concluded that there are distinct pH microdomains in discrete cellular regions of neuronal cell lines.