Ultrafast 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. 

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. 

Xu J, Toptygin D, Graver KJ, Albertini RA, Savtchenko RS, Meadow ND, Roseman S, Callis PR, Brand L, Knutson JR. Ultrafast fluorescence dynamics of tryptophan in the proteins monellin and iiaglc. J. Am. Chem. Soc. 2006 128 1214-1221. 

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. 

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... 

Ultrafast photoreaction dynamics in protein nanospaces (PNS) as revealed by fs fluorescence dynamics studies on photoactive yellow protein (PYP) and related systems... 

Abstract Ultrafast photoreactions in PNS of PYP have been studied by means of fs fluorescence up conversion method. Conclusions obtained are (a) Photoreaction in PNS (chromophore twisting) occurs from vibrationally unrelaxed fluorescence state and coherent oscillations in the fluorescence decay curves have been observed for the first time, (b) Comparative studies on fluorescence dynamics of mutants and w.-t. PYP have proved that the w.-t. PYP is best engineered for the ultrafast reaction, (c) The coherent oscillations in the fluorescence decay completely disappeared and the reaction was much slower in the denatured state, demonstrating the supremely important role of PNS for the photoreaction. 

Ultrafast fluorescence decay dynamics of w.-t. PYP and its mutants in PNS coupled with coherent vibrations... 

In order to examine further the effect of the PNS and PNS-chromophore interactions upon the ultrafast twisting reactions of the PYP chromophore, we are examining also the effect of the PNS environment on the fluorescence dynamics of the PYP analogues where the PYP chromophore is replaced with similar but a little different chromophores. Among those PYP analogues, we show here results of the fluorescence dynamics studies of those with (a) locked chromophore and (b) ferulic acid chromophore. 

Based on our fs fluorescence dynamics studies on w.-t PYP, various site-directed mutants, several PYP analogues and denatured PYP, we have demonstrated the supreme importance of the well-regulated PNS structure for the ultrafast and highly efficient photoinduced twisting of the chromophore leading to the isomerization. 

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... 

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... 

Bhasikuttan, A. C. Suzuki, M. Nakashima, S. Okada, T. Ultrafast fluorescence detection in tris(2,2 - bipyridine)ruthenium(II) complex in solution relaxation dynamics involving higher excited states. J. Am. Chem. Soc. 2002, 124, 8398-8405. 

The primary photoreactions in the PYP have been experimentally studied both in the protein and in solution environments. The initial structural change of PYP was directly observed by time-resolved x-ray crystallography. Ultrafast fluorescence spectroscopy was performed on the initial process of the photoreaction of PYP. It was shown that the photoisomerization reaction of the PYP chromophore is completed within 1 ps. The initial photoreaction processes were analyzed by using time-resolved spectroscopic data. Although the reaction of the PYP chromophore in solution environments has been studied by several groups, the characterization of the dynamics in these environments is not fully understood. 

Mataga, N., Chosrowjan, H., Shibata, Y., Tanaka, F., Nishina, Y., et al. Dynamics and mechanisms of ultrafast fluorescence quenching reactions of flavin chromophores in protein nanospace. J. Phys. Chem. B 104, 10667-10677 (2000)... 

M. Cho, S. J. Rosenthal, N. F. Scherer, L. D. Ziegler, and G. R. Fleming, J. Chem. Phys., 96, 5033 (1992). Ultrafast Solvent Dynamics Connection between Time Resolved Fluorescence and Optical Kerr Measurements. 

N. Tokunaga, E Imamoto, Y Kataoka, M. Ultrafast photoreactions in protein nanospaces as revealed by fs fluorescence dynamics measurements on photoactive yellow protein and related systems. Phys. Chem. Chem. Phys. 2003,5,2454-2460. 

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. 

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