Fluorescence up-conversion

It has been shown that photoexcitation of the guanine-cytosine (G-C) base pair leads to proton transfer [231], Watson-Crick (WC) base pairs have excited state lifetimes much shorter than other non-WC base pairs indicating once again that the natural occurring WC base pairs are more photostable than other alternative configurations [115, 118, 232-235], Much work has been done in the gas phase where many different base pair isomers exist. The ultrafast relaxation of the WC base pair has also been confirmed in solution using fluorescence up-conversion measurements [117]. 

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... 

The instruments that provide the best time resolution (about 100 femtoseconds) are based on fluorescence up-conversion. This very sophisticated and expensive technique will be described in Chapter 11. 

The technique of fluorescence up-conversion (see Chapter 11), allowing observations at the time-scale of picoseconds and femtoseconds, prompted a number of fundamental investigations on solvation dynamics that turned out to be quite complex (Barbara and Jarzeba, 1990) (see Box 7.1). 

With instruments based on fluorescence up-conversion (see Chapter 11) that offer the best time resolution (about 100 fs), such a fast inertial component was indeed detected in the fluorescence decayc,d). Using coumarin 153 as a solute (whose dipole moment increases from 6.5 to 15 D upon excitation), the inertial times of acetonitrile, dimethylformamide, dimethyl sulfoxide and benzonitrile were found to be 0.13, 0.20, 0.17 and 0.41 ps, respectively. ... 

Time-resolved fluorescence in the femtosecond time range fluorescence up-conversion technique... 

Chapter 6 described the current techniques employed in time-resolved fluorescence spectrocopy. The time resolution of these techniques ranges from a few picoseconds (streak cameras) to a few hundreds of picoseconds (single-photon timing with flash lamp excitation). The time resolution can be greatly improved by using the fluorescence up-conversion technique. 

Fig. n.1. Principles of fluorescence up-conversion. NLC nonlinear crystal DM dichroic mirror HW half-wave plate PM photomultiplier. 

Various ultrafast phenomena occuring in the femtosecond time-scale in the condensed phase have been studied by fluorescence up-conversion (for a review, see Mialocq and Gustavsson, 2001). As already mentioned in Chapter 7 (Box 7.1),... 

Fig. 11.2. Fluorescence up-conversion instrument. DM dichroic mirror HW halfwave plate GG420 Schott filter CCD video camera for the visual superposition of the...

Mialocq J.-C. and Gustavsson T. (2001) Investigation of Femtosecond Chemical Reactivity by Means of Fluorescence Up-Conversion, in Valeur B. and Brochon J. C. (Eds), New Trends in Fluorescence Spectroscopy. Applications to Chemical and Life Sciences, Springer-Verlag, Berlin, pp. 61-80. 

Finally, in Chapter 11 some advanced techniques are briefly described fluorescence up-conversion, fluorescence microscopy (confocal excitation, two-photon excitation, near-field optics, fluorescence lifetime imaging), fluorescence correlation spectroscopy, and single-molecule fluorescence spectroscopy. 

Other near-IR techniques that have been used to measure lifetimes, though not to the same extent as the aforementioned methods, include fluorescence up-conversion,(19 21) parametric amplification, 22 streak camera detection,(23) and two-photon excitation,1(24) The latter technique is particularly useful as it enables the greater penetration depth of near-IR radiation in organic matter to be used to obtain a well-defined region of excitation, e.g., in single cells or mammalian tissue. 

Contrary to the above-described detection methods, fluorescence up-conversion and optical Kerr gate techniques readily achieve picosecond/femtosecond time resolution (Ippen and Shank 1975 Shah 1988 Takeuchi and Tahara 1998), because they are in the pump-probe measurement, in principle. 

FIGURE 3.2 Principle of (a) fluorescence up-conversion and (b) optical Kerr gate. 

Here we first describe the ultrafast fluorescence microscope, which nses the fluorescence up-conversion method. This microscope simnltaneonsly achieves femtosecond time resolution and snbmicron space resolution (Fnjino and Tahara 2003, 2004). 

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)... 

FIGURE 3.4 Performance of the fluorescence up-conversion microscope, (a) Evaluation of the time-resolution with the 100 x objective lens , up-converted fluorescence -F-, the first derivative. By the fitting analysis, the time-resolution of the microscope was evaluated as 520 fs. (b) Evaluation of the transverse (XY) spatial resolution with the 100 x objective lens. A CCD image of the excitation pulses (inset) and the beam profile along the lateral (X) direction. By the fitting analysis, the transverse resolution was evaluated as 0.34 pm. (c d) Evaluation of the axial (Z) spatial resolution with the 100 x objective lens , up-converted fluorescence -I-, the first derivative. By fitting analysis on the first derivative coefficient, the axial resolution was evaluated as 1.1 pm with the 50 pm pinhole (c) and 5.3 pm without pinhole (d). (Rhodamine B, 2 x 10" mol dm in methanol, 600 nm.) (Erom Eujino, T. and Tahara, T., Appl Phys. B 79 145-151, 2004. Used with permission.)... 

Two-photon excitation can be used for the fluorescence up-conversion microscope, and high axial resolution was achieved without a pinhole in this case. Figure 3.5 shows the up-converted fluorescence from a coumarin 522B solution at a fluorescence wavelength of 520 nm observed in the same manner of Figure 3.4d without pinhole. In this measurement, a fundamental laser pulse at 800 nm was used for excitation. The axial resolution with two-photon excitation was evaluated to be 0.97 pm (FWHM) by fitting for the first derivative of the obtained data. This result indicates... 

The second example of the application of fluorescence up-conversion microscope is imaging of organic microcrystals based on ultrafast fluorescence dynamics (femtosecond fluorescence dynamics imaging) (Fujino et al. 2005a). In this measurement, the site-specific energy transfer rate in a tetracene-doped anthracene microcrystal was measured, and the crystal was visualized based on the observed local ultrafast dynamics. 

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... 

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