MRI Instrumentation Development: Construction of a Real-Time Fluorescence and Phosphorescence Lifetime Imaging 3D Microscope Based on a Novel Analog Acquisition Method

Cornell University, Ithaca NY

Investigators

Abstract

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). This instrument development project creates a new type of 3D fluorescence and phosphorescence lifetime imaging microscope based on a novel decay time measurement technique that operates in real-time. The decay time or lifetime is the time a molecule remains in an electronically excited state after absorbing a photon. Excited state lifetime measurements are insensitive to differential dye distributions and sample scattering and absorption differences, parameters that plague fluorescence intensity-only measurements, and can be used to measure ion concentrations, interactions between labeled or energy metabolism via NADH imaging. Phosphorescence lifetimes of certain compounds can be used for measuring oxygen concentrations. In fluorescence lifetime imaging microscopy (FLIM), the image pixel values reflect the fluorophore's lifetime rather than the intensity. In the project described here a new type of lifetime determination method is developed and applied in a FLIM system in which the lifetime image is displayed directly on the screen in real time enabling dramatically faster acquisition of lifetime images. The microscope construction will be carried out in the Developmental Resource for Biophysical Imaging Optoelectronics (DRBIO). Two instruments will be built, one designed for phosphorescence timescales (microsecond) which will be tested in projects requiring 3D-resolved oxygen imaging using oxygen-sensitive phosphors. A second instrument will be developed for fluorescence timescales (nanosecond) and tested a project that will utilize faster lifetime imaging for better elucidation of the regulation and interactions of molecular transcription initiators in vivo. The unique capabilities of the instrument - 3D-resolved lifetime imaging carried out 10-1000 times faster than possible on the currently available FLIM systems - will ensure that a number of future collaborative projects that will benefit from the system. The novel technique developed is not only applicable as an imaging modality, but can be applied in a number of analytical applications such as oxygen concentration sensing through an optical fiber. Design and fabrication of the instruments will provide an excellent opportunity for training graduate students and other researchers in optics, nonlinear optics, high speed electronics, biophysics and photophysics. In addition to the initial collaborating laboratories involved in the test bed experiments, the microscope will be available to researchers from around the world through DRBIO, a collaborative bioimaging center. Once developed and tested, the instrument design will be made commercially available by implementation on commercial laser scanning imaging systems.