Fluorescence depletion

The characterization of the laser pulse widths can be done with commercial autocorrelators or by a variety of other methods that can be found in the ultrafast laser literature. " For example, we have found it convenient to find time zero delay between the pump and probe laser beams in picosecond TR experiments by using fluorescence depletion of trans-stilbene. In this method, the time zero was ascertained by varying the optical delay between the pump and probe beams to a position where the depletion of the stilbene fluorescence was halfway to the maximum fluorescence depletion by the probe laser. The accuracy of the time zero measurement was estimated to be +0.5ps for 1.5ps laser pulses. A typical cross correlation time between the pump and probe pulses can also be measured by the fluorescence depletion method. 

Figure 2.30. Schematic energy-level diagrams depicting the pump-probe (a) OODR and (b) OODR-fluorescence depletion (OODR-FD) experiments. The y and z directions denote the polarization vectors of the various transitions. (Reprinted with permission from Ref. [49].)...

Third, one-photon excitation of the So (X) thiophosgene to the Si (A) state, followed by photodepletion of the S2 > So fluorescence (fluorescence depletion) by S (B, C) < So (A) excitation, by another photon, reveals the presence of the dark C state in the vicinity of the B state. The polarization as well as the inversion splitting patterns of the OODR-FD spectrum are consistent with the C state being the planar JB2 (na ) state predicted by an ab initio calculation. 

The structure of the 1 1 BA-H2O complex in its ground state has been determined by rotational coherence spectroscopy (RCS) using the fluorescence depletion scheme [74, 75], The empirical minimum-energy calculation supplemented the insufficient information obtained from the RCS measurement [74], In the structure giving the best fit to the RCS signal, the water molecule sits on the terminal aromatic ring of the one anthracene moiety. Thus the two anthracene moieties are stabilized by the water molecule in a different manner, and such asymmetry in the stabilization, i.e., symmetry breaking, facilitates the electron jump from one anthracene to the other [76]. 

In recent years, we have developed a two-color super-resolution laser scanning fluorescence microscope based on the up-conversion fluorescence depletion... 

Iketaki, Y., Watanabe, T., Ishiuchi, S., Sakai, M., Omatsu, T., Yamamoto, K. and Watanabe, T. (2003) Investigation of the fluorescence depletion process in the condensed phase application to a tryptophan aqueous solution. Chem. Phys. Lett., 372, 773-778. 

The above examples have all involved long-lived states leading to fluorescence from the upper excited state. In contrast, the fluorescence depletion method described above has been used to resolve shortlived levels of the E state of NO with line-widths as great as 10 cm (Ashfold et. al. 1986). In this case A - X fluorescence was excited by a two-photon transition with a blue dye laser, and e2e+ - a2e+ resonances were probed with a red dye laser. Even with these broad resonances it was possible to produce fluorescence dips of 50% - indeed care had to be taken to avoid power broadening because of the very large transition moment of the E-A inter-Rydberg transition. 

Fluorescence depletion meehanisms in superresolving STED microscopy. Chtan Phys Lett 442(4-6) 483-487... 

Femtosecond Time-Resolved Fluorescence Depletion Spectroscopy 321... 

A method, femtosecond time-resolved stimulated emission pumping (SEP) fluorescence depletion (FS TR SEP ED), has been developed to study the vibrational relaxation of electronic excited states of molecules (Figures 11.9 and 11.10) [31]. 

Kauffman, J.F., Cote, M.J., Smith, P.G., McDonald, J.D. Picosecond fluorescence depletion spectroscopy. 2. Intramolecular vibrational relaxation in the excited electronic state of fluorene. J. Chem. Phys. 90, 2874-2891 (1989)... 

Zhong, Q.H., Wang, Z.H., Sun, Y., Zhu, Q.H., Kong, F.N. Vibrational relaxation of dye molecules in solution studied by femtosecond time-resolved stimulated emission pumping fluorescence depletion. Chem. Phys. Lett. 248, 277-282 (1996)... 

Nagarajan, V., Parson, W. Femtosecond fluorescence depletion anisotropy application to the B850 antenna complex of Rhodobacter sphaeroides. J. Phys. Chem. B 104, 4010-4013 (2000)... 

Figure 3 A two-colour fluorescence depletion spectrum of one rovibronic line associated with the D n <- A n transition in SO. The two-colour excitation scheme used (upper right) is required because of the very short lifetime (100 fs) of the D P state. This results in the linewidth of 50 cm- shown in the spectrum.

Figfure 6. Sequence of measurements required for the fluorescence depletion technique (FRA). Shown is the complete data set for erythrosin-5 -isothiocyanate in a solid matrix of polymethyl- methacrylate. In traces (A) -(C) the parallel (to the actinic laser) [channels 1-512] and perpendicular [channels 513-1024] emission components are shown for (A) depletion signals (both lasers active) (B) excimer (actinic) laser only (C) steady-state fluorescence (i.e. with only the continuous wave argon laser) (D) polarized emission components after subtraction... 

As indicated earlier, only probes with relatively high values of isc suitable for phosphorescence emission measurements. The triplet state of fluorescein (see Table II) is not detectable by phosphorescence emission techniques but should provide a suitable probe for the fluorescence depletion technique (11,15,16). Figure 8 shows FRA data for the rotational diffusion at 6 C and in 98%... 

We wish to reveal the mechanism for the optimal dynamic discrimination between the very similar biochromophores riboflavin (RBF) and flavin mononucleotide (FMN) using optimally shaped laser fields. Our FISH simulations utilize experimentally optimized laser fields and show that the fluorescence depletion ratio between two molecules can be manipulated with such fields, eventually achieving discrimination between them. Moreover, these results validate for the first time the experimental optimal control technique applied on complex systems [63]. 

Fig. 17.6 Schematic illustration of the discrimination of FMN and RBF by fluorescence depletion. Excitation with a shaped UV laser pulse leads to transition from So to Sj state, as indicated by the light grey arrow. After a time-delay At during which dynamical processes take place, an unshaped IR pulse is applied. In the case of FMN (lefi part of the figure), this leads to transitions to higher excited states where irreversible processes such as ionization ctm occur (dark arrow), consequently the fluorescence gets depleted (crossed dark arrow). For RBF (right part of the figure), excitation to higher states is less favorable (crossed dark arrow), and fluorescence will remain stronger than in FMN (dark arrow). With differently shaped UV pulses, also the reverse situation is possible...

Fig. 17.9 Upper panel. Temporal structure of pulse 1 for maximization (left) and of pulse 2 for minimization (right) of the depletion ratio D(FMN)/D(RBF). Middle panel Ionized populations Pion of RBF (black) and FMN (red) due to pulse 1 (left) and pulse 2 (right). Lower panel Fluorescence depletion D of RBF (black) and FMN (red) due to pulse 1 (left) tmd pulse 2 (right) (Reprinted from Ref. [63], Copyright 2010 by the American Physical Society)...

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