Professor of Research, Biology
PO Box 400328
Biomedical Engineering, Biophysics
Imaging of Proteins in Living (or fixed) Cells and Tissues Using Light Microscopy Systems
I designed and developed different light microscopy imaging systems to investigate cellular signaling such as calcium, pH, protein-protein interactions and also diagnostic system in clinical imaging. The system includes: Digitized video microscopy, digital deconvolution, confocal, multi-photon excitation, fluorescence lifetime (FLIM), time-resolved, anisotropy and fluorescence resonance energy transfer (FRET) microscopy. My current research area of interest is energy-based FLIM-FRET Microscopy and Spectroscopy in the determination of when and where specific proteins associate with one-another in living cells and tissues.
Through the use of conventional fluorescence microscopy, proteins labeled with different fluorophores can be localized within fixed or living cell preparations. The fluorophores absorb light at one wavelength and emit light at another, longer wavelength. By using appropriate filters, it is possible to detect several different labeled proteins in the same preparation. However, the optical resolution of the light microscope limits determination of protein proximities to 0.2 micrometers. Resolving the relative proximities of proteins that exceeds the optical limit of the microscope is necessary to reveal the physical interactions between protein partners. This degree of spatial resolution can only be achieved in energy-based imaging using the technique of fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) microscopy. FRET is a process by which radiation-less transfer of energy occurs from a fluorophore in the excited state to an acceptor molecule in close proximity. The range over which resonance energy transfer can occur is limited to ~ 0.01 micrometers and the efficiency of energy transfer is extraordinarily sensitive to the distance between fluorophores. The measurement of FRET in the microscope provides a non-invasive approach to visualize the spatio-temporal dynamics of the interactions between protein partners in the living cell.
The fluorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state. Many currently available fluorescence microscopic techniques, such as confocal or multi-photon excitation, cannot provide detailed information about the organization and dynamics of complex cellular structures. In contrast, fluorescence lifetime imaging (FLIM) microscopy allows the measurement of dynamic events at very high temporal resolution and can monitor interactions between cellular components with very high spatial resolution as well. An important advantage of FLIM is that the absolute values of lifetimes are independent of the probe concentration, photobleaching, light scattering and the amount of excitation intensity. FLIM thus offers many opportunities for studying dynamic event of living cells.
A major obstacle to implementation of energy-based spectroscopy in living cells has been the lack of suitable methods for specifically labeling intracellular proteins with the appropriate fluorophores. The cloning of the jellyfish green fluorescent protein (GFP) and its expression in a wide variety of cell-types has proven this fluorescent protein to be a versatile marker for both gene expression and protein localization in living cells. When illuminated by blue light, the jellyfish GFP yields a bright green fluorescence that does not require any cofactors, substrates, or additional gene products. GFP retains its fluorescent properties when fused to other proteins, allowing energy-based microscopy to be used to visualize dynamic changes in protein localization in intact cells.