This is a proposal for instrumentation t perform measurements of fluorescent lifetimes using both the established technique single photon counting (SPU) and a new and novel method which we termed 'Time-Resolved Fluorescence Lifetime Imaging' (TRFLI). The system consists of a Ultrafast CW Nd: YAG pumped dye laser and a mode-locker, second and third harmonic generator units; an inverted epi- fluorescence microscope with infinity-corrected optic; microchannel-plate photomultipler tube; high-speed, gated image intensifier coupled fiberoptically to an integrating CCD camera, with necessary control units; digital delay pulse generator, delay units, time-to-amplitude converter with bias amplifier and multichannel analyzer; and on-stage specimen controllers for temperature and atmospheric gases ( O 2 & CO2). The requested laser has a minimum 1.3 ps pulse width, a repetition rate of 76 MHz and usable excitation wavelengths from 350-846 nm. Primary uses of this system will be to measure fluorescent lifetimes in living cells of fluorophores or fluorophore conjugated cellular components on the living cells. This information will then be used to determine with temporal and spatial resolution molecular interactions amongst cellular constituents, using resonance energy transfer and time resolved emission spectra, monitor membrane lipid order (fluidity) using time resolved emission anisotropy, detection of pathogenic organisms in clinical specimens and measurement of cytosolic free Ca2+ using Fura-2, Fluo-3, or Rhod-2 lifetime analysis. The use of Fluo-3 or Rhod-2 will also allow the use of caged compounds and laser scanning confocal microscopy. The TRFLI System will be a unique instrument integrating state-of-the-art advances in optical microscopy, low-light video detection and 2-dimensional lifetime image analysis and which does not exit anywhere else in the country.
