Ultrafast femtosecond fluorescence dynamics

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. 

Mataga, N., Chosrowjan, H., Taniguchi, S., Tanaka, F., Kido, N., et al. Femtosecond fluorescence dynamics of flavoproteins comparative studies on flavodoxin, its site-directed mutants, and riboflavin binding protein regarding ultrafast electron transfer in protein nanospaces. J. Phys. Chem. B 106, 8917-8920 (2002)... 

Underwood, D. E., Kippeny, T. and Rosenthal, S. J. (2001) Ultrafast carrier dynamics in CdSe nanocrystals determined by femtosecond fluorescence upconversion spectroscopy. /. Phys. Chem. B, 105,436-443. 

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... 

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]. 

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

Femtosecond spectroscopy has an ideal temporal resolution for the study of ultrafast water motions from femtosecond to picosecond time scales [33-36]. Femtosecond solvation dynamics is sensitive to both time and length scales and can be a good probe for protein hydration dynamics [16, 37-50]. Recent femtosecond studies by an extrinsic labeling of a protein with a dye molecule showed certain ultrafast water motions [37-42]. This kind of labeling usually relies on hydrophobic interactions, and the probe is typically located in the hydrophobic crevice. The resulting dynamics mostly reflects bound water behavior. The recent success of incorporating a synthetic fluorescent amino acid into the protein showed another way to probe protein electrostatic interactions [43, 48]. 

Certain chromophore systems are intrinsically predisposed for ultrafast single molecule microscopy. Among these, emitters coupled to metal surfaces stand out as exceptionally well-suited subjects. Numerous observations of substantial radiative rate enhancement at the surface or in the vicinity of the surface of a metal were reported. Radiative rate enhancements as large as 10 have been predicted for molecular fluorophores and for semiconductor quantum dots coupled to optimized nanoantennae.Such accelerated emission rates put these systems well within the reach of the emerging femtosecond microscopy techniques. As a result, we decided to apply the Kerr-gated microscope to study of fluorescence dynamics of individual core-shell quantum dots in contact with smooth and nanostructured metal surfaces. 

With site-directed mutation and femtosecond-resolved fluorescence methods, we have used tryptophan as an excellent local molecular reporter for studies of a series of ultrafast protein dynamics, which include intraprotein electron transfer [64-68] and energy transfer [61, 69], as well as protein hydration dynamics [70-74]. As an optical probe, all these ultrafast measurements require no potential quenching of excited-state tryptophan by neighboring protein residues or peptide bonds on the picosecond time scale. However, it is known that tryptophan fluorescence is readily quenched by various amino acid residues [75] and peptide bonds [76-78]. Intraprotein electron transfer from excited indole moiety to nearby electrophilic residue(s) was proposed to be the quenching... 

Among the best well-known examples of photostability after UV radiation, the ultrafast nonradiative decay observed in DNA/RNA nucleobases, has attracted most of the attention both from experimental and theoretical viewpoints [30], Since the quenched DNA fluorescence in nucleobase monomers at the room temperature was first reported [31] new advances have improved our knowledge on the dynamics of photoexcited DNA. Femtosecond pump-probe experiments in molecular beams have detected multi-exponential decay channels in the femtosecond (fs) and picosecond (ps) timescales for the isolated nucleobases [30, 32-34], The lack of strong solvent effects and similar ultrafast decays obtained for nucleosides and nucleotides suggest that ultrashort lifetimes of nucleobases are intrinsic molecular properties, intimately... 

Ultrafast fluorescence quenching dynamics were studied by the fluorescence-up-conversion method with femtosecond mode-locked laser systems. For the studies of oxazine dyes, a synchronously pumped hybrid mode-locked dye laser with group velocity... 

Theoretical papers on effects directly observable in the very short time regime are notable in this years collection. The theory of femtosecond pump-probe spectroscopy of ultrafast Internal conversion processes in polyatomic molecules has been developed using the behaviour of the excited pyrazine molecule as an example . The solvation dynamics for an ion pair in a polar solvent can now be examined by the time dependence of fluorescence and by direct observation of photoinduced charge... 

In fluid solvents at room temperature, spectral relaxation is usually comjdete prior to emission and occurs within abont 10 ps. This process is too rapid to be resolved with the usual instrumentation for TD or FD fluorescence. However, advances in laser technology and methods for ultrafast spectroscopy have resulted in an increasing interest in picosecond and femtosecond solvent dynaaiics. Becmise of the rapid timescale, the data on solvent dynamics are usually obtained using flurvescence upconversion. Hiis method is described in Section 4.7.C. lypical data are... 

Time-resolved femtosecond absorption, fluorescence, IR, and Raman spectroscopy elucidate the molecular structure evolution during ultrafast chemical reactions [7-11]. The technique provides in real time direct insight into the structural dynamics of various systems including photoisomerization. In this chapter, we briefly describe modem methods of studying photochemical and photophysical processes that have been employed or can be employed in the stilbene photophysics and photochemistry. 

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