Fluorescence lifetime imaging of the pH distributions in living cells
AdvisorLakowicz, Joseph R.
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AbstractIntracellular pH distribution is a critical physiological determinant of diverse cellular functions such as gene expression, vesicular transportations, and lysosomal degradation. Inhibition of the pH regulatory mechanisms in astrocytes and osteoclasts was shown to induce serious pathological defects. As a consequence, accurate and efficient imaging techniques to follow the pH distributions in living cells are essential to biomedical research. Fluorescence-based detections have become popular techniques in determining intracellular pH. The general detection principles include wavelength shifts, intensity-ratio changes, and lifetime changes. The recent progress of time-resolved detection has made fluorescence lifetime imaging microscopy a potential pH-imaging tool. However, reports focusing on lifetime-based pH imaging in organelles are scarce. The objective of this dissertation is to use the frequency-domain FLIM technique to image the pH distributions in cytosol and in vesicular organelles lysosomes. In addition, we evaluated the performance of FLIM in correlating intracellular pH variations with specific pH regulatory processes. Based on the previous lifetime characterization results, Carboxy-SNAFL2 was used for cytosolic pH imaging. We are able to image the cytosolic pH in different adherent cells and follow the transient cytosolic pH variations in living CHO cells. DM-NERF dextrans, OG-514 carboxylic acid dextrans, and LysoSensor DND-160 were used to image the pH distributions in acidic compartments since they showed appropriate low pH working range. The resting lysosomal pH values determined from 3T3 fibroblasts are between pH 4.5 and 4.9. It was also possible to follow the significant alkalization of the lysosomal pH after the treatments of proton-pump inhibitors and ionophores with FLIM. These imaging results demonstrate that our FLIM apparatus is able to provide comparable pH resolution and sensitivity to those obtained from intensity ratio imaging measurements and from other lifetime imaging techniques. In order to resolve the cellular structures at the sub-micron level, our present FLIM apparatus needs to be upgraded with the pseudo-confocal capability. We expect the future installation of multi-photon multi-focal microscopy to the FLIM instrumentation will greatly improve the z-axis resolution of the lifetime images. The future plans are to resolve the possible pH gradients existing in cytosol and describe the pH evolution in endosomal vesicles during endocytosis processes.
DescriptionUniversity of Maryland, Baltimore. Molecular Biology and Biochemistry. Ph.D. 2001
fluorescence lifetime imaging microscopy