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Instruments




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)

  • Argon Ion (Coherent) mode locked laser, 1W at 514 nm, fwhm = 120 ps.
  • Ti:Sapphire femtosecond laser system (Spectra Physics), 750-920 nm, can be frequency doubled (375 - 460 nm) or frequency tripled (250 - 310 nm); fwhm = 90 fs
  • Ti:Sapphire, regenerative amplifier, optical parametric amplifier system (Coherent) 120 fs fwhm; tunable from UV-IR.
  • Argon Ion air cooled, CW, 488, 514.5 nm.
  • HeCd air cooled, CW, 442 nm (Liconix)
  • HeCd air cooled, CW, 325 nm (Liconix)
  • Modulated CW laser diodes and light emitting diodes

Dye Lasers

  • Rhodamine 6G cavity-dumped dye laser, 560-620 nm, 280-310 nm, after doubling, fwhm = 5 ps (with saturable absorber if needed)
  • Pyridine 1 and pyridine 2 cavity-dumped dye lasers, 680-760 mn, 340-380 nm after doubling, fwhm = 7 ps
  • DCM cavity-dumped dye laser, 620-680 nm, 310-340 nm after doubling, fwhm = 7 ps

Photodetectors for TCSPC

  • MCP-PMT, Hamamatsu R2809, with a red-sensitive photocathode, 60 ps fwhm
  • PMT, Philips XP2020, 500 ps fwhm

Photodetectors for FD

  • MCP-PMT, Hamamatsu R2566, 6 µ, for FD measurements to 10 GHz
  • PMT, Hamamatsu R928, for FD measurements to 300 MHz
  • A red-sensitive 6 µ R2566, for FD measurements to 10 GHz

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.

  • 50/50 beamsplitter: cross-correlations of the APD photoelectrons to observe fast intensity fluctuations in the range of 1-10 microseconds.
  • Polarization beam cube: fluorescence anisotropy studies.
  • Dichroic mirrors: fluorescence resonance energy transfer studies.

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.

Witec 300s NSOM/AFM  

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

  • TCSPC with a MCP-PMT and a ps laser or fs excitation source
  • FD up to 10 GHz using the harmonic-content of a ps or fs pulse train
  • TCSPC with a laser and fast PMT
  • FD from 3 KHz to 400 MHz with a modulated cw source

Local Network

Domain Name: umbi.umd.edu

Operating Systems: MS-DOS, Windows '98, 2000, XP, Linux

Software:

Data Analysis: 

  • MathCad
  • MatLab
  • Origin
  • Slidewriter
  • Microsoft Visual Studio
  • LabView

Data Publishing:  

  • Adobe Photoshop
  • Adobe Acrobat
  • Corel WordPerfect
  • Microsoft Office
  • Microsoft Power Point
  • ChemDraw
 

COMPUTER SPECIFICATIONS

  • CFS Server (Web, E-mail, FTP)
  • PC's running under Windows'98, Windows 2000, Windows XP operating system.

    Network
     
  • Data transfer between computers possible via Internet (HTTP, FTP). Data and/or programs are also available on diskettes (MS-DOS, LS120, Zip-Drive), or CD/DVD-RW, external USB/FW hard drives.
  • Terminals adjacent to instrument room

SUPPORTING CAPABILITIES

  • Steady state emission spectra (SLM 4800 Spectrofluorometer and SLM AB-2)
  • Absorption diode array (190 nm - 1100 nm) spectrophotometer. (Hewlett Packard)
  • Linear dichroism and transition moment determination
  • Lab space adjacent to instrument room
  • Temperature control, -60 to +90°C
  • Ultracentrifuge (Beckman L5-65B), 65,000 rpm
  • Gas pressure cell to 100 atm, and a 2 kbar hydrostatic pressure cell