Pulsed excitation/gated detection

Time-gated detection offers the possibility to suppress background signals correlated with the excitation pulse. Direct and multiple scattered excitation light as well as Raman scattering reaches the detector at t 0, and can be effectively suppressed by opening the first gate a few hundred picoseconds after t = 0. 

In Fig. 3.5A a comparison between time-gated detection and TCSPC is shown. The time-gated detection system was based on four 2 ns wide gates. The first gate opened about 0.5 ns after the peak of the excitation pulse from a pulsed diode laser. The TCSPC trace was recorded using 1024 channels of 34.5 ps width. The specimen consisted of a piece of fluorescent plastic with a lifetime of about 3.8 ns. In order to compare the results, approximately 1700-1800 counts were recorded in both experiments. The lifetimes obtained with TG and TCSPC amounted to 3.85 0.2 ns and 3.80 0.2 ns respectively, see Fig. 3.5B. Both techniques yield comparable lifetime estimations and statistical errors. 

Phosphorescence spectra (uncorrected, front face) were recorded on a Perkin-Elmer LS-5 fluorescence spectrometer using a pulsed excitation source ( 10 ps) and gated detection. The instrument was controlled by a P-E 3600 data station. The samples were typically excited at 313 nm using the instrument s monochromator and an additional interference filter. Excitation and emission bandpasses were 2 nm. Typically the emission spectra were recorded using a 50 ps delay following excitation and a 20 ps gate. The samples were contained in cells made of 3x7 mm2 Suprasil tubing, under a continuous stream of N2, 02 or 02/N2 mixtures of known composition. 

Fig. 28 Schematic view of the Zeiss Plate Vision instrument which is state-of-the art for ultra-high throughput screening (uHTS) for drug discovery. The instrument resembles a 96-well parallel microscope the light of a excitation source (Xe-lamp or pulsed laser) is expanded to illuminate a microtiter plate. The excitation is structured into 96 channels by a mini-lens array (MLA) and focused into the well with a detection volume of < 100 nL. All 96 channels are read simultaneously by a gated, intensifed CCD. With this fast detector and the pulsed laser excitation, the instrument can be used to carry out miniaturized, 96 parallel lifetime measurements in microtiter plate format with nanosecond time resolution or time-gated detection [190]...

Figure 4. Time-resolved fluorescence decay curves of two differently concentrated R-6G solutions. The initial sharp peak is due to the excitation pulse. The time window during which fluorescence counts are accumulated in time-gate detection is also shown. (Adopted from [30].)...

Direct observation of phosphorescence from conjugated polymers has been achieved by the application of gated detection techniques. In these techniques the detection window of an intensified CCD is delayed with respect to the excitation laser pulse. Therefore the detector is blocked during the intense prompt fluorescence caused by the conjugated polymer and able to detect the delayed emission that is usually orders of magnitude lower than prompt fluorescence. Spectrally resolved detection allows for the observation of the shape and energetic position of the delayed emission. By varying the width of the detection window... 

Raman spectra can be recorded in times as short as several nanoseconds using pulsed laser excitation/gated array detection techniques. This capability is important for the identification of transient species during fast chemical reactions or for characterizing structural failure modes in thin films exposed to heat or intense electromagnetic fields. An existing system... 

TCSPC, intensity decays were measured using stroboscopic or pulse sampling methods. The basic idea is to sample tfie intensity decay repetitively following pulsed excitation (Figure 4.25). The detection gato is dtsi aced across the intensity decay until the entire decay is measured. In fact, the first TD lifetime instruments used gated detection to sample the intensity decay. ... 

Another coherent technique that can be combined with gated detection is levelcrossing spectroscopy (Sect. 7.1). If the upper atomic levels are excited by a pulsed... 

TR-FRET assays are, by definition, assays applying gated detection. Due to their relatively wide use in drug discovery, practically all plate reader vendors have adapted their instmments to read TR-FRET assays too. Both xenon flash lamps and nitrogen lasers are applied in the plate readers to create short-lived and intense excitation pulses at the area of 330-340 nm. 

---