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 al., 2010). 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 Eclipse 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 chemical reactions.