Developmental Resource for Biophysical Imaging and Opto- Electronics

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The global mission of the "Developmental Resource for Biophysical Imaging and Opto-electronics" consists of the creation, development, facilitation and application of new instrumentation technologies for the visualization and measurement of dynamic cellular and molecular processes with the ultimate objective of solving fundamental problems in biological research. A renaissance in light microscopy, driven by advances in laser physics, nonlinear optics, microelectronics and computer technology has engendered powerful new optical and microprobe technologies. Instrumentation research and development has recently produced nonlinear laser microscopy based on two photon excitation (TPE) which occurs by simultaneous molecular absorption of two low energy photons to create a high energy excitation that generates fluorescence or photochemistry. This Resource invention was motivated by a need for instantaneous spatially resolved photochemistry as well as three dimensionally resolved, background-free fluorescence microscopy. Much of this research program aims to realize the potential of this invention and to establish its methodology and utility through exploratory biophysical applications. We propose developments aimed particularly at dynamical measurements in living nervous system preparations such as neurotransmitter micropharmacology by point photolysis of caged molecules, fast (video rate) ratiometric calcium imaging, and optimization of methods for thick, highly scattering biological specimens. Resource developments include nanometer spatial displacement measurements, nanometer scale tracking of individual molecules, and most recently a picoNewton-sensitivity optical force probe whose operation in a scanning mode provides surface mapping by Optical Force Microscopy (OFM). These instruments are enabling new Resource biophysics research on cell surface receptor dynamics, sensory transduction, cell motility mechanisms, and second messen ger signalling mechanisms. Resource development of multiplexed instrumentation that accommodates cmmbinations of these probes, atomic force microscopy (for larger forces) and fluorescence imaging and molecular tracking on living cells is proposed. Using these novel technologies, research objectives are the in vivo measurement and mapping of picoNewton interaction forces, dynamics of ligand-receptor pairs, cell surface compliances and individual intermolecular forces. Specific Research Objectives: (1) Technology of two photon laser scanning microscopy (TPLSM) -- Design and develop TPLSM research workstations capable of fluorescence decay time imaging, video rate ratiometric imaging, programmable caged neurotransmitter release and single particle tracking. (2) Exploratory applications of laser microscopy and two photon excitation -- Develop new instrumentation techniques to exploit the advantages of TPLSM, in particular (a) techniques for thick, strongly scattering, living preparations, (b) localized fast cage activation for micropharmacology, (c) specific applications to nervous system preparations, and (d) specific applications to plant tissue. (3) Photochemistry and photophysics -- laser excitation in the cellular environment -- Determine mechanisms and controls of photochemical processes that ultimately limit the effectiveness of both one and two photon fluorescence microscopy in living cells. Understand operative mechanisms of one and two photon photobleaching, cellular photodamage, photoablation and the kinetics of cage activation. Apply photoablation and cage activation to biological systems. (4) Nonlinear laser microscopy measurements of molecular mobility -- Develop instrumentation and techniques for 3-d resolved molecular mobility measurements using two photon excited fluorescence photobleaching recovery (FPR), Fluorescence Activation Redistribution (FAR), and Fluorescence Correlation Spectroscopy (FCS). Apply to photoregulation of second messenger responses, receptor-secretion coupling, and neurotransmitter release. (5) Measurement of Protein Dynamics and Folding by Fluorescence Correlation Spectroscopy (FCS) -- Apply FCS aided by spatially localized two photon excitation as a new fast probe of protein folding and dynamics. (6) Nanometer microscopy and picoNewton forces -- Develop multimode nanometer instrumentation and techniques for scanning cell surface receptor interactions and for practical measurements of force-velocity relations down to the picoNewton level. Combine Optical Force Microscopy (OFM) with optical tweezers manipulation, nanometer molecular tracking, fluorescence imaging, atomic force microscopy AFM), and possible near field fluorescence excitation.