Bacterial Protein Expression Core
Cell Biology and Imaging Core
EM Crystallography Core
EM Tomography Core
Eukaryotic Protein Expression Core
Fluorescence Spectroscopy Core
Protein Interactions Core
Protein NMR Spectroscopy Core
RNA Structure and Dynamics Core
Tissue EM Core
Virus Imaging Core
X-ray Crystallography Core
Fluorescence Spectroscopy Core
Director: David Millar, PhD
The Fluorescence Spectroscopy Core provides state-of-the-art resources for fluorescence spectroscopic measurements, including fast-time resolved and single-molecule methods. We are equipped to measure fluorescence excitation and emission spectra, polarization anisotropy, fluorescence lifetimes and FRET, in both bulk and single-molecule formats.
Instrumentation and Capablities
Single-molecule Fluorescence Detection
Two separate systems are available, one for TIRF-based imaging of surface-immobilized molecules and a second system for solution-based FRET and FCS measurements. The TIRF imaging system is based on an Axiovert 200 inverted microscope, equipped with a Zeiss 1.45 N.A. objective, a dual-view optical splitter and an electron multiplying CCD camera (Andor). Separate solid-state lasers are used for blue (488 nm), green (532 nm) and red (634 nm) excitation. A prism-based TIR illumination system is used to excite surface-immobilized molecules. TIRF images and movies are acquired using the associated camera software or custom IDL software. Further data analysis is performed using custom MATLAB software, generating FRET efficiency trajectories (time traces) and histograms. The solution-based system is based on an Axiovert 200 inverted microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a Zeiss oil immersion objective (N.A. 1.3) for excitation and collection of emitted fluorescence. Argon-ion and helium-neon lasers (Melles-Griot Lasers, Carlsbad, CA) are used for continuous excitation in the visible spectral range, while a femtosecond titanium:sapphire laser (Coherent Mira) is used for 2-photon excitation. Dichroic mirrors and interference filters are used to block excitation light and separate the fluorescence into two colors (donor and acceptor) for ratiometric FRET measurements. Avalanche photodiode detectors allow highly efficient fluorescence detection with sub-microsecond time resolution. Up to four independent detection channels can be recorded simultaneously using a computer-based signal acquisition card. A digital correlator (ALV 6010) is used for on-line auto- and cross-correlation measurements. Custom software is used for electronics control and data processing and allows for a variable bin time. Results are presented in the form of single-pair FRET efficiency histograms and/or fluorescence auto- and cross-correlation functions.
Time-resolved Fluorescence Studies
A picosecond mode-locked titanium: sapphire laser (Coherent Mira) equipped with pulse-picker and frequency doubler/tripler is used for excitation (tunable from 275 to 330 nm, 400 to 520 nm, 750 to 1050nm). A Hamamatsu micro channel plate photomultiplier and time-correlated single photon system (Ortec) are used to acquire fluorescence decay profiles. Fluorescence lifetimes and associated amplitudes are obtained by fitting the decay curves with multi-exponential functions.
Steady-state and Stopped-flow Fluorescence Studies
The Core has a SLM8100 spectrofluorimeter equipped with T-format optics, polarizers, photon counting detection and stopped-flow rapid mixing cell. The detection electronics and instrument software have been updated with the Phoenix package (ISS).
Dedicated Single-Molecule TIRF microscope for Characterizing Cellular Complexes
During the current funding cycle, we will purchase an Ixon X3 Multiplying CCD camera and a Zeiss C-Apochromat Water Immersion Objective. This instrumentation will allow us to build a new, dedicated single-molecule TIRF microscope that will be used to characterize complexes that we purify directly from mammalian cell extracts.
RNP Assembly at the Single-molecule Level. An RRE fragment capable of binding 4 Rev monomers was immobilized on a quartz surface, fluorescently labeled Rev (grey oval and star) was introduced, and individual complexes monitored over time in a single-molecule TIRF microscope (left). Numbers above each segment of the trajectory indicate the number of bound Rev molecules. Analysis of multiple trajectories yields rate constants for each step of assembly (right). This work was performed in the Millar lab, in a collaboration with the Williamson lab.