Zeiss 2-photon (2P) LSM780 laser scanning confocal microscope

Levi, Moshe

Abstract

We are requesting funds to purchase a Carl Zeiss 780 NLO Confocal microscope, which we will couple to a Coherent Chameleon Ultra femto-second Ti: sapphire laser and ISS fast-fluorescence lifetime imaging detector. This system will be the only inverted two-photon microscope generally available at the Anschutz Medical Campus and will be the only fluorescence lifetime imaging system available to the research community in the Greater Denver area. The Zeiss LSM 780 NLO will have tremendous impact on NIH funded research as it is capable of performing Fluorescence Lifetime Imaging Microscopy (FLIM), Two-Photon Excitation (TPE) and Second Harmonic Generation (SHG) Microscopy, and also Hyperspectral Imaging. Fluorescence Lifetime Imaging Microscopy (FLIM): FLIM is becoming a very powerful technique for in vivo spatiotemporal imaging of dynamic changes in mitochondrial metabolism and oxidative stress. In cancer biology the recently reemerging original concept of Warburg effect is also related to alterations in glycolysis versus mitochondrial oxidative phosphorylation. Dynamic imaging of metabolism is achieved in part through monitoring changes in NAD(P)H and FAD lifetimes. FLIM is also a very sensitive technique to perform Foster (or Fluorescence) Resonance Energy Transfer (FRET), a powerful technique to monitor protein-protein interactions of biological interest that occur within a distance of 10 nm or less. Unlike the widely used intensity techniques that are limited by spectral bleed through and spectral overlaps, changes in lifetime can detect real changes in protein-protein interactions without suffering from the limitations associated with the intensity based techniques. Two Photon Excitation (TPE) and Second Harmonic Generation (SHG) Microscopy: SHG is a very powerful technique that allows for label-free imaging of fibrillary collagens. Fibrillary collagens resulting in tissue fibrosis occurs in many pathophysiological conditions associated with tissue fibrosis including kidney disease, liver disease, lung disease, and heart disease. In cancer tissues the so called epithelial to mesenchymal transformation is also associated with accumulation of fibrillary extracellular matrix proteins. In contrast in advanced atherosclerosis activation of tissue matrix metalloproteinases can result in localized decreased in matrix proteins. Spectral Unmixing: This new and improved modality available with the Zeiss LSM780 is a powerful technique that will allow simultaneous imaging of several fluorophores corresponding to several proteins, DNA molecules, or lipids of interest without the complication of bleed through that we have encountered in the past.