Dynamic fluorescence

Lee, J., O Kane, D. J., and Gibson, B. G. (1989). Bioluminescence spectral and fluorescence dynamics study of the interaction of lumazine protein with the intermediates of bacterial luciferase bioluminescence. Biochemistry 28 4263-4271. 

Figure 21.5 Fluorescence dynamics of Au(0) i, for excitation at 395 nm, and emission at 570 nm. The corresponding numerical fits to the data are indicated by the thin solid lines. The residuals of the fit are shown at the top of graph.

By altering the detector setup, data can be collected from fluorescence intensity or fluorescence lifetime measurements. For TPM data acquisition for home-built systems, the SimFCS computer program (Laboratory for Fluorescence Dynamics, University of California at Irvine) is a free resource available to researchers. 

D. Application 2 Femtosecond Fluorescence Dynamics Imaging EROM Tetracene-Doped Anthracene Microcrystal... 

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. 

FIGURE 3.7 (a) The CCD camera image of the tetracene-doped anthracene microcrystal used for the femtosecond fluorescence dynamics imaging, (b) The dynamics image obtained from the region indicated by a broken rectangle in (a) (excitation 400 nm fluorescence 530 nm). (From Fujino, T., Fujima, T., and Tahara, T., J. Phys. Chem. B 109 15327-15331, 2005. 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... 

Fujino, T, Fujima, T, and Tahara, T. 2005. Femtosecond fluorescence dynamics imaging using a fluorescence up-conversion microscope. J. Phys. Chem. B 109 15327. 

Krishnan, R. V., Biener, E., Zhang, 1. H., Heckel, R., and Herman, B. 2003. Probing snbtle fluorescence dynamics in cellular proteins by streak camara based fluorescence hfetime imaging microscopy. Appl. Phys. Lett. 83 4658. 

Fluorescence dynamics of coumarin C522 in water and in cyclodextrin cavity... 

The host-guest p-cyclodextrin-C522 complex formation was determined based on fluorescence blue shift as a function of the increasing p-cyclodextrin concentration from 10 6 to 10 2 M. Similar result was observed for coumarin C6 [4] and this blue shift was considered along with anisotropy results as a proof of the host-guest formation. Time-resolved fluorescence spectroscopy was utilized to differentiate between fluorescence dynamics of... 

Fig, 2. Left and right parts show the fluorescence dynamics of C522 in water and in p-cyclodextrin aqueous solution for the fluorescence wavelengths of 500 (squares), 520 (circles), and 540 (triangles) nm, respectively. The experimental and simulation data are scatter and line, respectively. 

Time-resolved fluorescence of coumarin C522 was determined in water and in host-guest complex with p-cyclodextrin, representing free aqueous and cavity restricted environments, respectively. Experimental fluorescence clearly showed faster dynamics in a case of water. The time parameters of monoexponential fit for water and p-cyclodextrin at 500 nm and 520 nm were determined to be 1.37 ps and 2.02 ps, and 2.97 ps and 7.14 ps, respectively. Multi-mode Brownian oscillator model, as an attempt to simulate the solvation dynamics, supported these fluorescence dynamics results. 

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. 

Fig. 2. The values of S2 decay rate constant A, ( ) estimated from the S2 fluorescence decay times and the rate constant of the S, state formation Xp (O) by S2 excitation obtained from the S, fluorescence dynamics for ZP-I series in (a) Tol and (b) THF solutions plotted against -AGCS values.

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. 

From such a viewpoint, we are examining primary processes of photoreactions of PYP [1] which functions as a blue light photoreceptor for a negative phototaxis of the purple sulfur bacterium Ectothiorhodospira halophila, some FP s [2] and Rh [3] by means of the fs fluorescence up-conversion measurements. In this article, we will discuss our latest results of fs fluorescence dynamics studies on PYP, because PYP is very stable for repeated irradiation which induces photocycles so that the very accurate experimental results can be obtained rather easily and also the preparation of the site-directed mutants as well as the PYP analogues with modified chromophores are rather easy. However, before that, we will summarize briefly results of our previous investigations. 

Comparative studies on fs fluorescence dynamics of w.-t. (wild type) PYP and site-directed mutants... 

We have also examined the fs-ps fluorescence dynamics of various mutants, the preparation of which were described elsewhere [4], In these mutants, the hydrogen bonding networks surrounding the chromophore are weakened or partially broken or the structure of the protein where the chromophore is linked by thioester bond becomes looser, leading to the more disordered PNS structure. 

The fluorescence dynamics measurements were carried out with fluorescence up-conversion apparatus [1(b)] based on Ti Sapphire laser (820 nm, 800 mW, 76 MHz, -65 fs). The fwhm of the instrumental response was -110 fs. 

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. 

Fig. 4. Fluorescence dynamics studies on PYP analogues effects of the PNS - chromophore interactions.

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. 

Fig. 2c. It can be seen that at 530 nm, the fluorescence decays mono-exponentially with the fluorescence lifetime of 3.24 ns. The rise of the emission seen below 50 ps in the corresponding FlUp data is obviously not resolved here. In contrast, the TCSP data at 450 nm is described by a triple-exponential decay whose dominant component has a correlation time well below the time resolution. This component is obviously equivalent to the fluorescence decay observed in the FlUp experiment. A minor contribution has a correlation time of about 3.2 ns and reflects again the fluorescence lifetime that was also detected at 530 nm. The most characteristic component at 450 nm however has a time constant of about 300 ps. It is important to emphasize that this 300 ps decay does not have a rising counterpart when emission near the maximum of the stationary fluorescence spectrum is recorded. In other words, the above mentioned mirror image correspondence of the fluorescence dynamics between 450 nm and 530 nm holds only on time scales shorter than 20 ps. Finally, in contrast to picosecond time scales, the anisotropy deduced from the TCSPC data displays a pronounced decay. This decay is reminiscent of the rotational diffusion of the entire protein indicating that the optical chromophore is rigidly embedded in the core of the 6-barrel protein.

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