Fluorescence up-conversion technique

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. 

Fluorescence Up-Conversion Technique. A completely different approach from the ones described above is used in the up-conversion technique. The development of this technique originates in the desire... 

Formation of a stable protein-DNA complex involves the rearrangement of water molecules and release of counter ions and water molecules to the bulk. Zewail and co-workers have used the time-resolved fluorescence up-conversion technique to explore the dynamics of the histone-DNA complex formation and the participation of hydration water in the stability and specificity of the recognition process [9]. This important study established the contribution from the entropic gain due to the release of hydration water (often termed dynamically ordered water) to the bulk. 

Another class of porphyrins, substituted by 3,5-di-i-butylphenyl groups at the a and Y meso positions, has recently been studied using the fs fluorescence up-conversion technique [128], Following excitation into the Sj state, the zinc metalloporphyrin species undergoes an Sj-Sj internal conversion with a time constant of 150 fs before intramolecular vibrational relaxation takes place in the Q state with a time constant of 600 fs. In the case of the free-base porphyrin, the system relaxes from the B state to the Qy state within 40 fs, followed by internal conversion between the Qy and states in 90 fs. Subsequently, IVR occurs in the state in 1.5 ps before the porphyrin returns to its ground state in 12 ns. 

The time-resolved emission experiment was performed with the fluorescence up-conversion technique. (For details see Ref. 11). Excitation parameters were Xexc = 865 nm (Rb. sphaeroides), tp 200 fs, less than 10 % of the RC absorb a photon. Up-conversion process collinear type II phase matching in a 1mm BBO crystal up-converted fluorescence emission 910-930 nm width of the instrumental response function cr 400 fs. 

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). 

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. 

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... 

A more direct way of exploring the dynamics of polar solvent in the presence of a solute is by the optical excitation of a solute to an intramolecular charge transfer state and then observing the time-dependent fluorescence, as shown in Fig. 1.7 [47, 52]. To follow the time-dependent fluorescence at short times, faster than the usual fluorescent lifetime of nanoseconds, lasers plus an up-conversion technique were used. A quantity frequently measured is the dynamic Stokes shift S(r),... 

Both TCSPC and frequency-domain fluorimetry are limited in time resolution by the response of available detectors, typically >25 ps. For cases in which higher time resolution is needed, fluorescence up-conversion can be used (22). This technique uses short laser pulses (usually sub-picosecond) both to excite the sample and to resolve the fluorescence decay. Fluorescence collected from the sample is directed through a material with nonlinear optical properties. A portion of the laser pulse is used to gate the fluorescence by sum frequency generation. The fluorescence is up-converted to the sum frequency only when the gate pulse is present in the nonlinear material. The up-converted signal is detected. The resolution of the experiment therefore depends only on the laser pulse widths and not on the response time of the detectors. As a result, fluorescence can be resolved on the 100-fs time scale. For a recent application of fluorescence up-conversion to proteins, see Reference 23. 

In principle the analysis of up-conversion data is performed in a similar fashion as in the SPT technique. With the advent of very short laser pulses (femtoseconds), the up-conversion technique is becoming more and more popular for the measurement of very short fluorescence decays. It is not, however, a technique that is easily adapted as a general method in the life sciences. 

Figure 9.17 Time evolution of the fluorescence quenching in a 20% PPPV/PC blend by an electric field of 1.7 x 10 V cm- and T = 70 K. Data recording involved the technique of fluorescence up-conversion. The full curve is theoretical. (From Kersting, R. et al., Phys. Rev. Lett, 73, 1440, 1994. With permission.)...

Watanabe, T., Iketaki, Y., Omatsu, T., Yamamoto, K., Sakai, M. and Fujii, M. (2003) Two-point-separation in superresolution fluorescence microscope based on up-conversion fluorescence depletion technique. Opt. Express, 11, 3271-3276. 

An excellent alternative to the streak camera approach is fluorescence time resolution by the "up-conversion" method, which we describe in detail below. In the simplest form of this technique, non-linear optical methods are employed to essentially construct a picosecond "shutter" or "gate". In most applications a single emission datum is acquired for each laser pulse, i.e. I(t=constant, X=constant). By repeating the experiment at different delay times, kinetic traces can be acquired that are of comparable quality to those obtained by streak camera methods (16-17). Alternatively, if delay time is held constant but X is scanned, high quality emission spectra can be obtained (3). 

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