Quantitative photochemical applications of the multiphoton laser-scanning microscope

This work explores the use of the Multiphoton Laser Scanning Microscope (MPLSM) to quantitatively study biophysical processes in living systems with high spatial and temporal resolution. Two novel multiphoton excitation (MPE)-based photolysis techniques are presented, the first of which is Multiphoton Fluorescence Photobleaching Recovery (MP-FPR). This technique measures the diffusion coefficient of fluorescently labeled molecules in solution with three-dimensional resolution. A small volume of the molecules is bleached via MPE and the subsequent recovery to equilibrium of the fluorescence signal in the bleached area is monitored, yielding the diffusion coefficient of the labeled molecule. The analysis required to derive diffusion coefficients is presented, and the parameters contributing to random and systematic errors in the technique are discussed and quantified. The accuracy of MP-FPR is demonstrated by the measurement of a known diffusion coefficient, and the applicability of this technique to in vivo measurements is also shown. The second MPE-based photolysis technique described in this work is quantitative MPE calcium uncaging. Calcium cages are buffers that drastically lower their affinity for calcium upon photoexcitation, thereby rapidly releasing the ion. A novel method to measure the uncaging action cross section of calcium cages is described, and is applied to three cages. I present the mathematical tools necessary to use these cross sections in order to estimate the total amount of calcium released with a given MPE dose, as well as the spatial and temporal distribution of the released calcium under various buffering conditions. To explore the applicability of quantitative MPE calcium uncaging to in vivo experiments, the behavior of calcium in dendritic spines, as generated by an action potential (the event of neuronal action), is explored. Using the MPLSM, the behavior of the calcium in single spines after an action potential is found to be governed by diffusion between the spine and nearby dendrite, in parallel with calcium extrusion mechanisms. The limitations of quantitative MPE calcium uncaging in thick living samples is also explored.