Fargoland is a unique time-resolved fluorometer that processes luminescence
signals induced by nanosecond-wide laser pulses. It uses a passively Q-switched
YAG microchip laser (532 nm, 355 nm, and 266 nm), with ~1 mJ pulse energy and
extremely uniform pulse shape and intensity, a high-sensitivity photomultiplier,
and a transient digitizer with high time resolution (8 Gs/sec) and linearity, to
directly record the fluorescence decay waveform. From a typical
fluorescent-labeled biological sample, this instrument records a reproducible
waveform with high precision (S/N > 100) following a single laser pulse every
0.1 ms, 100,000 times faster than the best commercial single-photon-counting
fluorometer (Muretta et
). Signal averaging for 0.1 s (1000 pulses) increases S/N by a
factor of about 30.
Fargoland operates under three modes coupled with respective accessories,
providing maximum flexibility for diverse research needs.
- Time-resolved fluorometer, TRF. This mode
is equipped with a Peltier temperature-controlled cuvette holder with magnetic
stirring and four open windows for fluorescence detection. The emission
wavelength is selected using an interference filter, and time-resolved
fluorescence anisotropy can be acquired using a computer-controlled emission
polarizer. This instrument can be used to resolve complex lifetime and distance
distributions via TR-FRET (Agafonov
et al., 2009; Kast et
al., 2010). A time-correlated single-photon counting (TCSPC) module is also
available, using pulsed diode lasers. Data acquisition is much slower (A typical
experiment takes 10s or longer.), but additional wavelengths are available: 375
nm, 405 nm, and 485 nm.
- Time-resolved fluorescence spectrometer, TRFS. The accessory is
also equipped with a Peltier temperature-controlled cuvette holder. The emission
monochromator of the Varian
instrument permits the acquisition of time-resolved fluorescence spectra over
the entire emission spectrum.
- Transient time-resolved fluorometer, (TR)2F.
This mode utilizes a two-syringe stopped-flow module (Bio-Logic, Claix, France,
1.5 ms dead time), and a custom designed fluorescence collection system.It is
used for double-kinetic experiments in which a submillisecond fluorescence
waveform is acquired every 0.1 ms. This instrument is uniquely capable of
analyzing transient structural kinetics in FRET-labeled proteins (Nesmelov et al., 2011).
Our state-of-art data analysis program, FargoFit, is designed to analyze
virtually an unlimited number of waveforms by global least-squares minimization,
using a wide range of fitting models, including light emission, quenching,
fluorescence resonance energy transfer, rotational diffusion, and transient
Location: 1-150 Nils Hasselmo Hall