Laser fluorescence detector experiment

Estimate the fluorescence rate (number of fluorescence photons/s) on the Na transition 5s -> 3p, obtained in the Doppler-free free-photon experiment of Fig. 7.28, when a single-mode dye laser is timed to v/2 of the transition 3s 5s in a cell with a Na density of n = 10 cm. The laser power is P = 100 mW, the beam is focused to the beam waist wo = 10 cm and a length L = 1 cm around the focus is imaged with a collection efficiency of 5% onto the fluorescence detector. 

A laser beam highly focused by a microscope into a solution of fluorescent molecules defines the open illuminated sample volume in a typical FCS experiment. The microscope collects the fluorescence emitted by the molecules in the small illuminated region and transmits it to a sensitive detector such as a photomultiplier or an avalanche photodiode. The detected intensity fluctuates as molecules diffuse into or out of the illuminated volume or as the molecules within the volume undergo chemical reactions that enhance or diminish their fluorescence (Fig. 1). The measured fluorescence at time t,F(t), is proportional to the number of molecules in the illuminated volume weighted by the... 

The experiment is performed with a spectrofluorometer similar to the ones used for linear fluorescence and quantum yield measurements (Sect. 2.1). The excitation, instead of a regular lamp, is done using femtosecond pulses, and the detector (usually a photomultiplier tube or an avalanche photodiode) must either have a very low dark current (usually true for UV-VIS detectors but not for the NIR), or to be gated at the laser repetition rate. Figure 11 shows a simplified schematic for the 2PF technique. 

In conventional chip experiments, fluorescence scanners are used for chip read-out. In the case of laser scanners, HeNe lasers are used as excitation sources and photomultiplier tubes as detectors, whereas CCD-based scanners use white light sources. The optical system can be confocal or non-confocal. Standard biochip experiments are performed using two fluorescent labels as... 

The first fluorescence correlation spectroscopy experiments were carried out several decades ago,62 64 but the general use of the technique was made possible with the introduction of lasers with high beam quality and long-term temporal stability, low noise detectors, and high-quality microscope objectives with high numeric apertures.58,63 The most common set-up is using a confocal inverted epi-fluorescence... 

Both TCSPC and frequency-domain fluorimetry are limited in time resolution by the response of available detectors, typically >25 ps. For cases in which higher time resolution is needed, fluorescence up-conversion can be used (22). This technique uses short laser pulses (usually sub-picosecond) both to excite the sample and to resolve the fluorescence decay. Fluorescence collected from the sample is directed through a material with nonlinear optical properties. A portion of the laser pulse is used to gate the fluorescence by sum frequency generation. The fluorescence is up-converted to the sum frequency only when the gate pulse is present in the nonlinear material. The up-converted signal is detected. The resolution of the experiment therefore depends only on the laser pulse widths and not on the response time of the detectors. As a result, fluorescence can be resolved on the 100-fs time scale. For a recent application of fluorescence up-conversion to proteins, see Reference 23. 

The transient absorption method utilized in the experiments reported here is the transient holographic grating technique(7,10). In the transient grating experiment, a pair of polarized excitation pulses is used to create the anisotropic distribution of excited state transition dipoles. The motions of the polymer backbone are monitored by a probe pulse which enters the sample at some chosen time interval after the excitation pulses and probes the orientational distribution of the transition dipoles at that time. By changing the time delay between the excitation and probe pulses, the orientation autocorrelation function of a transition dipole rigidly associated with a backbone bond can be determined. In the present context, the major advantage of the transient grating measurement in relation to typical fluorescence measurements is the fast time resolution (- 50 psec in these experiments). In transient absorption techniques the time resolution is limited by laser pulse widths and not by the speed of electronic detectors. Fast time resolution is necessary for the experiments reported here because of the sub-nanosecond time scales for local motions in very flexible polymers such as polyisoprene. 

The molecular jet of molecules is crossed with a tunable dye laser and the laser-induced fluorescence is collected with a lens and focused on a PMT detector (Fig. 5). In the original experiments, a standard pulsed dye laser was used to match the 10-Hz duty cycle of the pulsed valve and the pulsed Nd YAG vaporization laser. Although this approach provides a high S/N ratio and wide spectral coverage, the resolution is limited by the laser line width of typically 0.5cm-1 (no etalon) to 0.05cm-1 with an etalon. 

Spectroscopy of single molecules is based on fluorescence correlation, photoncounting histograms, or burst-integrated-lifetime techniques. Each case requires recording not only the times of the photons in the laser period, but also their absolute time. Modem time-resolved single molecule techniques therefore use almost exclusively the FIFO (time-tag) mode of TCSPC. The FIFO mode records all information about each individual photon, i.e. the time in the laser pulse sequence (micro time), the time from the start of the experiment (macro time), and the number of the detector that detected the photon (see Sect. 3.6, page 43). 

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