|
|
|
|
The Center for Fluorescence Spectroscopy (CFS) provides
access to state-of-the-art time-domain (TD) and frequency-domain (FD)
fluorescence instrumentation for time-resolved studies of biological
macromolecules. The excitation sources are cavity-dumped and
frequency-doubled ps dye lasers, or a Ti:Sapphire laser. Time-correlated
single photon counting (TCSPC) is accomplished with a microchannel plate (MCP)-PMT,
to provide an instrument response function near 60 ps. Frequency-domain
measurements are possible up to 10 GHz using the Center's FD instrument, and
a high speed MCP-PMT. Available excitation wavelengths range from UV to NIR.
For less demanding applications modulated cw lasers (for FD) is available. A
unique capability of the CFS will be the ability to collect and analyze both
TD and FD data for the same samples, and in the future, simultaneous
dual-domain (DD) analysis of the data. A Ti:Sapphire laser is now available
for two- and three-photon excitation. INSTRUMENT SPECIFICATIONS EXCITATION SOURCES (PRIMARY)
DYE LASERS
PHOTODETECTORS FOR TCSPC
PHOTODETECTORS FOR FD
ISS Alba fluorescence correlation spectrometer (FCS). This FCS instrument is connected to an Olympus IX71 inverted microscope. A 60x 1.2NA water immersion objective is used to create the excitation volume, typically ~1 femtoliter. An argon ion laser is used for an excitation source (488 or 514.5 nm). The FCS instrument uses two Perkin Elmer avalanche photodiodes with dark counts < 10 cps. A number of different optical filters are used to direct the fluorescence emissions towards the detectors.
PerkinElmer Proscan DNA array reader. This instrument can read two-color DNA arrays printed on standard 1”x4” glass slides. The lower resolution limit is 5 microns. Two solid state lasers are used for excitations. The 532 nm laser is good for exciting dyes such as Cy3, Tetramethyl rhodamine and Texas Red. The 640 laser can be used to excite Cy5 or other near infrared absorbing dyes. LI-COR Odyssey near infrared flatbed scanner. This system was two excitation/detection channels, optimized for 700 nm and 800 nm fluorescence. The 25x25 cm surface can accommodate a variety of samples formats, for example microtiter plates, gels, microarrays, tissue sections and small animals. The lower resolution limit is 20 microns. PicoQuant single-molecule scanner. This instrument is used mainly for fluorescence studies of molecules immobilized on coverslips. Two APDs are used for sensitive photon detection. An x-y axis piezo scanner stage rasters the sample which is mounted on an Olympus IX-71 microscope. Two primary excitation sources are pulsed diode lasers at 474 nm and 638 nm. An integrated time-correlated single-photon counting board enables lifetime measurements on single fluorophores, including fluorescence lifetime imaging microscopy (FLIM). Additionally, the PicoQuant can be used for FCS. Lab-built single-molecule scanner .This instrument was built at the Center for Fluorescence Spectroscopy. The microscope can image in several modes. In confocal mode, an x-y axis piezo scanner is used to raster the sample across the objective. Fluorescence can be spectrally resolved onto two APDs for dual-color or FRET imaging. The system utilizes microfluidic cells so measurements can be performed on immobilized molecules, or diffusing molecules (FCS). The fluorescence emissions can be imaged onto a spectrograph to obtain single-molecule spectra. Alternatively, objective-type total internal reflection (TIR) can be used to excite surface-bound molecules. With TIR, the excitation field decays exponentially from the surface (1/e value of ~200 nm) which greatly reduces scattering from the bulk solution. An electron-multiplied CCD is used to image TIR illuminated samples. EXPERIMENTAL CAPABILITIES
COMPUTER SPECIFICATIONS
SUPPORTING CAPABILITIES
|