Laser-induced fluorescence fluorescent molecular probes

The high sample demands and low-throughput of LC-MS methods have led to the creation of a capillary electrophoresis (CE) platform for ABPP [48]. Proteomes are labeled with a fluorescent probe, digested with trypsin, and enriched with antifluorophore antibody resins. Use of CE coupled with laser-induced fluorescence (LIF) detection to analyze the enriched peptides resulted in far superior resolution to ID SDS-PAGE, particularly for enzymes that share similar molecular masses. Sensitivity limits of 0.05-0.1 pmol/mg proteome, negligible sample requirements (—0.01—0.1 pg proteome), and the ability to perform rapid CE runs in parallel with 96-channel instruments, make CE-based ABPP a potentially powerful technique. One drawback is that the identities of the probe-labeled proteins are not immediately apparent, and correlated LC-MS experiments must be performed to assign protein identities to the peaks on the CE readout. 

Laser-induced fluorescence (LIF) depends on the absorption of a photon to a real molecular state, and is therefore a much more sensitive technique, capable of detection of sub-part-per-billion concentrations. Thus, this is the most suitable for measurement of those minor species which are the transient intermediates in the reaction network. Here a tunable laser is required, as well as an electronic absorption system falling in an appropriate wavelength region serendipitously, many of the important transient species have band systems which are suitably located for application of LIF probing. The ability to sensitively detect transitions originating from electronically as well as vibrationally excited levels of a number of molecules offers the possibility of inquiring into the participation of non-equilibrium chemistry in combustion processes. 

The ground electronic state of 139La160 is X2S+ audits electronic spectrum involving the excited B2Y,1 has been studied by Doppler-free laser-induced fluorescence by Bacis, Collomb and Bessis [85] and by Bernard and Sibai [86]. Both states have therefore been well characterised and the system is ideal for radiofrequency/optical double resonance, as described by Childs, Goodman, Goodman and Young [87]. They used a collimated molecular beam, with the laser pump/probe technique described elsewhere in this chapter. 

Combustion processes are driven by energy-releasing chemical reactions. Detailed knowledge of the chemical kinetics of these individual reactive steps is required input to combustion models. For more than a decade, elementary gas-phase reaction kinetics has been successfully studied with the flash photolysis/resonance fluorescence technique (1-8). Typically, following broadband photolysis of a molecular precursor, reactant decays have been measured under pseudo-first-order kinetic conditions with cw resonance lamp excitation of free radical fluorescence. Increased utilization of laser probes in kinetic studies is exemplified by the recent pulsed-laser photolysis/pulsed-laser-induced fluorescence experiments of McDonald, Lin and coworkers (9-13). 

The experimental arrangement for such femtosecond experiments is exhibited in Fig. 10.14. The output pulses from a femtosecond pulse laser (Sect. 6.1.5) are focused by the same lens into the molecular beam. The probe pulses are sent through a variable optical-delay line and the absorption a (At) of the probe pulse as a function of the delay time At is monitored via the laser-induced fluorescence. Cutoff Alters suppress scattered laser light. 

In a beam the molecules can travel over considerable distances in the vacuum chamber without experiencing collisions. Depletion of the population of a specific level by means of optical pumping at one place can be probed at another place by observing the laser induced fluorescence. If the level is repopulated by some process in the region between the two places the laser induced fluorescence increases. This Rabi type experiment with optical state selection has been applied by Childs and Goodman for the detection of molecular hfs transitions in the radiofre-... 

In order to avoid such secondary OODR signals, sub-Doppler excitation under collision-free conditions has to be realized. This can be achieved in a collimated molecular beam that is intersected by the two lasers LI and L2 either at two different positions z and zi (Fig-10.18) or with two overlapping laser beams (Fig. 10.19). In the first arrangement the probe laser-induced fluorescence /fi( 2) can be imaged separately onto the detector, and chopping of the pump laser LI with phase-sensitive detection of yields... 

The diatomic yttrium halides have been the topic of both ab initio and experimental studies. Fischell et al. (1980) have studied the excitation spectra of the YCl diatomic molecule using the laser-induced fluorescence (LIF) method. More recently, Xin et al. (1991) have studied the B ri-X system of YCl in high resolution. The rotational analysis of the observed bands has yielded very accurate molecular constants for the X and B states of YCl. Shirley et al. (1990) have studied the molecular-beam optical Stark spectrum of the B n(t = 0)-X (t = 0) band system of YF. The permanent dipole moment and the magnetic hyperfine parameter a for the B n state have been determined as 2.96(4) D and 146.8(3) MHz, respectively. The dipole moment of the X S state was determined as 1.82(8)D. More recently, Shirley et al. (1991) have employed the molecular-beam millimeter-wave optical pump-probe spectroscopy to study pure rotational transitions of the YF ground state. This study has yielded improved ground-state rotational constants as B = 8683.65(1) MHz and D = 0.0079(2)-MHz, respectively. 

In the present article we give an overview of recent work carried out in our laboratory in order to study microscopic details of bimolecular gas phase reactions at the molecular level using the laser photolysis / laser-induced fluorescence (LP/LIF) pump-and-probe technique. In particular, we will focus on the following three- and four-atom reactions ... 

Transient Infrared Absorption (TRISP) and laser-induced fluorescence. Because the CJ temperatures are only 2000-3000 K, most of the molecular products are in the ground electronic state. Emission spectroscopy looks selectively at only a few extraordinary molecules which are scarcely representative of most of the products. Infrared absorption, on the other hand is ideal for probing the vibrotational states of the ground state molecules, and the fast response time of TRISP makes it ideal for detonations. The technique has not been applied extensively and is difficult to implement, but our preliminary attempts have shown that we can do it with the proper laser apparatus. Broadband CARS is an alternative approach if the instrumental difficulties of TRISP cannot be overcome. 

Figure 7.3 Apparatus for flash studies of fast reactions in molecular beams (1). Schematic drawing of a molecular-beam flash apparatus. The pump and probe pulses (see text. Section 4.2.4.3) are produced by a tunable dye laser, a beam-splitter, and a delay line, not shown in the figure (see Figure 7.4). The two pulses are recombined by the beam-splitter BS and sent coaxially into the molecular-beam apparatus. A supersonic jet of (e.g.) argon gas is generated by expanding the gas through a nozzle into a vacuum chamber (not shown), for time-of-flight measurements. This apparams was used in smdies of the dissociation of iodine molecules and subsequent recombination (see below. Section 7.3.4.1 and Ref. [17]). Monitoring was by laser-induced fluorescence (LIF).

Figure 7.11 Iodine dissociation and recombination, 21 (2). Sub-picosecond transient in an argon cluster. Laser-induced-fluorescence transient after excitation of iodine molecules to the A state by a pump pulse of 614 nm at a series of pump-probe delay times. Upper panel Molecular-dynamics simulation for an hAr44 cluster with an initial temperature of 30 K prohe wavelength 307 nm. Lower panel Experimental transient from an iodine-argon molecular beam. The simulation reproduces the initial peak a at time zero, the decrease and recovery during the first picosecond b, c the subsequent slower rise, and some of the observed modulations, indicated by vertical arrows. See text. After Ref. [17,b],...

This optical-optical double-resonance technique has already been used for other Doppler-free techniques [10.25], such as polarization spectroscopy (see Sect.10.3). Its applications to molecular beams has, however, the following advantages compared to spectroscopy in gas cells. When the chopped pump laser periodically depletes the level E. and populates level Ej, there are two relaxation mechanisms in gas cells which may transfer the population modulation to other levels. These are collision processes and laser-induced fluorescence (see Fig.8.39). The neighboring levels therefore also show a modulation and the modulated excitation spectrum induced by the probe laser includes all lines which are excited from those levels. If several absorption lines overlap within their Doppler width, the pump laser simultaneously excites several upper states and also partly depletes several lower levels. 

---