Time-resolved fluorescence depletion

Femtosecond Time-Resolved Fluorescence Depletion Spectroscopy 321... 

Doxombicin binds readily to ceU membranes, changing their structure and function. The targets of doxombicin binding are compounds with a negative charge, of which the most extensively studied is the phospholipid cardiolipin (Pollakis et ah, 1983). Cardiolipin occurs in high concentrations in the inner mitochondrial membrane, where it is required for full activity of cytochrome c oxidase. In a recent study (Das and Mazumdar, 2000), the interaction between cytochrome c oxidase and cardiohpin in the presence of doxombicin was analysed, and the results of pico-second time-resolved fluorescence depolarization showed that the cardiohpin layer was depleted due to complexation with the dmg. 

In this section, we discuss methods that detect the time delay td between excitation of a fluorophore and arrival of a fluorescence photon. The distribution of times constitutes the fluorescence decay profile of the fluorophore. The average time lag between the excitation event and the emission is the fluorescence lifetime y of the fluorophore. The fluorescence decay contains information about dynamic processes that deplete the excited state (Fig. 2a). In time-resolved fluorescence experiments, the fluorescence decay is measured to gain information about these processes. 

First, we will describe the fluorescence kinetics after excitation with an ultrafast excitation pulse that can be approximated by a 5-pulse in the absence of nonradiative processes that could deplete the excited state (idealized case of time-resolved fluorescence decay measurements) [11, 12]. In a system of equivalent fluorophores (embedded in a homogeneous medium and interacting equally with the microenvironment), all the excited molecules have the same probability of emission of a photon but, due to the stochastic nature of the spontaneous emission, only the relationships concerning large numbers of fluorophores can be formulated. It is obvious that the number of photons released per unit time (rate of emission, or fluorescence intensity, F (xdNp/dt) in the system without competing nonradiative processes equals the total rate of de-excitation (depletion of the excited state), —dN/dt, which is proportional to the number of fluorophores excited at a given time, N t). Hence, we can write — dN/dt = N(t), where is the rate constant of... 

Fig. 3 The outline of fast dynamic processes that proceed at rates comparable with that of the radiative depletion of the excited state and can be studied by time-resolved fluorescence techniques (TRF) the rate of the polymer chain dynamics (vibrational motion and relaxation) strongly overlaps that of electronic relaxation and can be studied by TRF. Adapted from Springer, Self Organized Nanostructures of Amphiphilic Block Copolymers I, 241, 2011, 187-249, figure 3, [2], Copyright 2011. With kind permission from Springer Science and Business Media...

The monochromatic laser beam is split into two beams 1 and 2, both of which cross the molecular beam perpendicularly but at different locations = A and 2 = 8 (see Fig.10.10). When the laser frequency is tuned to a molecular transition (vIj, JIj - V, J ), the molecules passing through the pump beam 1 are optically pumped and there are nearly no molecules left in the depleted level (vIjjJ . ). This means that the fluorescence excited by the probe beam 2 is very low. If the pump beam 1 is interrupted for a short time interval At (e.g., by a fast mechanical chopper) the molecules can pass during this interval At without being pumped. Because of their different velocities they reach the probe beam 2 at different times t = L/v, where L = 22 - The time-resolved fluorescence intensity induced by the cw probe beam therefore reflects the velocity distribution of molecules in the level (vV,JV). 

In a case where dissociation is faster than the decay of < >OH, (ki k(f) + k(nr)) the emission of < )OH will decline very rapidly, due to the depletion of population by the dissociation reaction. The steady-state reflection of this rapid dissociation will be a very weak emission of the OH species. Slow dissociation of < >OH, or rapid recombination of the excited anion with the proton (H+-fc ]>Ai) will enhance the emission of OH (observed by steady-state fluorescence) and prolong the decay time of < >OH as observed by time-resolved measurements. 

If the first laser beam is interrupted at the time to for a time interval At that is short compared to the transit time T = z2 — Z )/v (this can be realized by a Pock-els cell or a fast mechanical chopper), a pulse of molecules in level /) can pass the pump region without being depleted. Because of their velocity distribution, the different molecules reach zi at different times t = to- -T. The time-resolved detection of the fluorescence intensity /fi(0 induced by the second, noninterrupted laser beam yields the distribution n T) = n(Az/v), which can be converted by a Fourier transformation into the velocity distribution n(v). Figure 4.13 shows as an example the velocity distribution of Na atoms and Na2 molecules in a sodium beam in the intermediate range between effusive and supersonic conditions. If the molecules Na2 had been formed in the reservoir before the expansion, one would expect the relation Up(Na) = V2up(Na2) because the mass m(Na2) = 2m(Na). The result of Fig. 4.13 proves that the Na2 molecules have a larger most probable velocity Up. This implies that most of the dimers are formed during the adiabatic expansion [410]. 

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]. 

If the first laser beam is interrupted at the time to for a time interval Ar that is short compared to the transit time T = z2 z )/v (this can be realized by a Pockels cell or a fast mechanical chopper), a pulse of molecules in level /) can pass the pump region without being depleted. Because of their velocity distribution, the different molecules reach Z2 at different times t to + T. The time-resolved detection of the fluorescence intensity /fi(0 induced by the second, noninterrupted laser beam yields the distribution... 

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)... 

---