Femtosecond fluorescence up-conversion

Peon J, Zewail AH (2001) DNA/RNA nucleotides and nucleosides direct measurement of excited-state lifetimes by femtosecond fluorescence up-conversion. Chem Phys Lett 348 255... 

FIGURE 3.3 Schematic diagram of the femtosecond fluorescence up-conversion microscope. (From Fnjino, T. and Tahara, T. J. Phys. Chem. B 107 5120-5122, 2003. Used with permission)... 

As described in the previous section, the femtosecond fluorescence up-conversion microscope enabled us to visualize microscopic samples based on position-depen-dent ultrafast fluorescence dynamics. However, in the imaging measurements using the fluorescence up-conversion microscope, XY scanning was necessary as when using FLIM systems. To achieve non-scanning measurements of time-resolved fluorescence images, we developed another time-resolved fluorescence microscope. 

The time-resolved techniques that are usually used for FLIM are based on electronic-basis detection methods such as the time-correlated single photon counting or streak camera. Therefore, the time resolution of the FLIM system has been limited by several tens of picoseconds. However, fluorescence microscopy has the potential to provide much more information if we can observe the fluorescence dynamics in a microscopic region with higher time resolution. Given this background, we developed two types of ultrafast time-resolved fluorescence microscopes, i.e., the femtosecond fluorescence up-conversion microscope and the... 

The measurements of the fluorescence dynamics were made by a femtosecond fluorescence up-conversion apparatus similar to that described elsewhere [2], The fivhm of the instrumental response was 110 fs. The polarization axis of the pump pulse was set at 54.7° with respect to the probe to suppress the anisotropy effects. 

Ultrafast photophysics of the protonated Schiff base of retinal in alcohols studied by femtosecond fluorescence up-conversion... 

Transient absorption experiments have shown that all of the major DNA and RNA nucleosides have fluorescence lifetimes of less than one picosecond [2—4], and that covalently modified bases [5], and even individual tautomers [6], differ dramatically in their excited-state dynamics. Femtosecond fluorescence up-conversion studies have also shown that the lowest singlet excited states of monomeric bases, nucleosides, and nucleotides decay by ultrafast internal conversion [7-9]. As discussed elsewhere [2], solvent effects on the fluorescence lifetimes are quite modest, and no evidence has been found to date to support excited-state proton transfer as a decay mechanism. These observations have focused attention on the possibility of internal conversion via one or more conical intersections. Recently, computational studies have succeeded in locating conical intersections on the excited state potential energy surfaces of several isolated nucleobases [10-12]. 

Sample preparation was given elsewhere [2]. Femtosecond fluorescence upconversion and picosecond time-correlated single-photon-counting set-ups were employed for the measurement of the fluorescence transients. The system response (FWHM) of the femtosecond fluorescence up-conversion and time-correlated single-photon-counting setups are 280 fs and 16 ps, respectively [3] The measured transients were fitted to multiexponential functions convoluted with the system response function. After deconvolution the time resolution was 100 fs. In the upconversion experiments, excitation was at 350 nm, the transients were measured from 420 nm upto 680 nm. Experiments were performed under magic angle conditions (to remove the fluorescence intensity effects of rotational motions of the probed molecules), as well as under polarization conditions in order to obtain the time evolution of the fluorescence anisotropy. 

The femtosecond fluorescence up-conversion setup has been described elsewhere [13,14]. Briefly, a second harmonic (SH) of a home-made chromium-forsterite femtosecond laser tunable from 610 to 660 nm was used to excite the sample (Fig.2) [14]. The pulse duration of the SH pulses was about 50 fs at the full width at half maximum (FWHM). We were successful in the cavity-dumping operation of this laser [14] and kept the repetition rate as low as 4 MHz. Reduction of the repetition rate was necessary to avoid multiple hits of the same location of the sample as small as possible. The excitation intensity, controlled by a neutral density filter before the sample cell, was (0.5-l)xl012 photons/cm2/pulse. Special care was taken to work at the lowest excitation light intensity so that the effect of the exciton-exciton annihilation process was negligible. 

Femtosecond fluorescence up-conversion microscopy a new method to study ultrafast dynamics in microstructures... 

Fig. 1. The CCD camera image of the a-perylene microcrystal used for the femtosecond fluorescence up-conversion microscopy...

Thus, we attempted to apply another experimental approach, femtosecond fluorescence up-conversion spectroscopy, to detect the excited-state dynamics of bovine rhodopsin in real time [51,52]. In addition, we also measured the protonat-ed Schiff bases of 11-ds-retinal and 11-ds-locked 5-membered ring retinal in methanol solution, which provided experimental evidence about how protein contributes to efficient isomerization [53]. 

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