Laboratory for Fluorescence Dynamics

Laboratory for Fluorescence Dynamics

Research Emphasis

Biological fluorescence spectroscopy has been undergoing a transition from studies of extracted biomolecules—such as proteins, nucleic acid polymers, lipid assemblies, and other more complex systems in large-volume cuvettes—to microscopic samples with an ultimate resolution at the single-molecule level. In this environment, the research emphasis of the Laboratory for Fluorescence Dynamics is geared toward the development of technologies that facilitate the transition to microscope-based systems. The confluence of multiphoton laser excitation techniques and fluorescence microscopy applications has led to rapid advances in imaging techniques as well as in related methods, such as fluorescence correlation spectroscopy (FCS). FCS offers high spatial resolution and observation of motional dynamics in single molecules identified from correlation functions or photon-counting histogram analysis methods. Other research areas include fluorescence in turbid media (such as tissue), global analysis software that allows tests of models (such as quenching, lifetime heterogeneity, and energy transfer), and interferometry.

Current Research

New methods and instrumentation to study cellular structure and function; imaging of tissue, particularly skin; and macromolecular dynamics and interactions. Examples of cellular studies include green fluorescent protein (GFP)-conjugates, receptor (high-density lipoprotein) function, ion distribution, and photosynthesis in algae. Other studies relate to membrane fluidity and domains (in large unilamellar vesicles), membrane-associated enzymes, protein-lipid interactions, protein folding, DNA-protein interactions, and vascular injury. Monitoring of physiologic function of tissue, based on variations in spectroscopic properties (such as fluorescence lifetime-based detection in medical endoscopy).

Resource Capabilities

Instruments

Time-resolved and steady-state fluorescence instrumentation: The frequency domain (phase/modulation; 300 MHz) instrumentation with laser excitation (titanium sapphire, argon ion, or mode-locked Nd/YAG pumped dye laser) covers the ultraviolet to the near-infrared regions. The photon-counting scanning fluorometers record emission/excitation and polarization spectra and kinetics. The sample compartments of the lifetime and spectral instruments accommodate high pressure, gas quenching, and other types of vessels in addition to thermostatting conventional cuvettes.

Light/fluorescence microscopy: For the study of cells and other microscopic structures, several laser-based multiphoton excitation systems (Ti:sapphire; 700B1000 nm) are coupled to light microscopes with fluorescence capabilities. These systems allow for imaging (in plane and three-dimensionally), particle tracking, and a variety of image-capture approaches based on fluorescence spectra, intensity, polarization, generalized polarization, and lifetime. In addition, these systems are compatible with measurements of fluorescence correlation spectroscopy. Software has been developed to process the images and the FCS data (auto- and cross-correlation and photon-counting histograms).

The laboratory houses a data analysis center, spectroscopy laboratories, microscopy laboratories, a wet chemistry laboratory, and a tissue culture facility.