Near fluorescence lifetime

The vast majority of single-molecule optical experiments employ one-photon excited spontaneous fluorescence as the spectroscopic observable because of its relative simplicity and inlierently high sensitivity. Many molecules fluoresce with quantum yields near unity, and spontaneous fluorescence lifetimes for chromophores with large oscillator strengths are a few nanoseconds, implying that with a sufficiently intense excitation source a single... 

Ambrose W P, Goodwin P M, Martin J C and Keller R A 1994 Alterations of single-molecule fluorescence lifetimes in near-field optical microscopy Science 265 364-7... 

Dunn R C, Holtom G R, Mets L and Xie X S 1994 Near-field fluorescence imaging and fluorescence lifetime measurement of light harvesting complexes in intact photosynthetic membranes J. Chem. Phys. 98 3094-8... 

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

Bloch, S., Lesage, F., McIntosh, L., Gandjbakhche, A., Liang, K. and Achilefu, S. (2005). Whole-body fluorescence lifetime imaging of a tumor-targeted near-infrared molecular probe in mice. J. Biomed. Opt. 10, 054003. 

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. 

Until recently there were comparatively few reports of fluorescence lifetime studies of dye molecules in the near-IR, but this situation has changed rapidly. The fluorescence lifetimes of near-IR emitting dyes such as carbocyanines, porphyrins, oxazines, and xanthenes, are usually in the nanosecond region, consistent with the high oscillator strength of the Si-So transition in such compounds. 

Farrens and Song<40) have replaced the original spark source with a picosecond diode laser in a multiplexed dual wavelength T-formatfluorometer.(41)With an overall instrumental response width of ca. 300 psec full-width half-maximum (FWHM), near-IR fluorescence lifetimes as low as 75 psec in the case of l,l -diethyl-4,4 carbo-cyanine iodide (DCI) (excitation 660 nm) and decay components as low as 48 psec in the case of 124 kDa oat phytochrome (excitation 752 nm) were reported. 

The opportunities for near-IR fluorescence sensors are of course not only limited to analytical chemistry. Physical parameters such as temperature can also be measured. For example, Grattan and Palmer have used the fluorescence lifetime quenching of neodymium glass fluorescence at 1054 nm, excited at 810 nm with a gallium-alumi-... 

The first fluorescence lifetime measurements reported using near-IR spark source excitation where performed in the authors laboratory using the all-metal coaxial... 

D. J. S. Birch, G. Hungerford and R. E. Imhof, Near-infrared spark source excitation for fluorescence lifetime measurements, Rev. Sci. Instrum. 62, 2405-2408 (1991). 

The pK of tyrosine explains the absence of measurable excited-state proton transfer in water. The pK is the negative logarithm of the ratio of the deprotonation and the bimolecular reprotonation rates. Since reprotonation is diffusion-controlled, this rate will be the same for tyrosine and 2-naphthol. The difference of nearly two in their respective pK values means that the excited-state deprotonation rate of tyrosine is nearly two orders of magnitude slower than that of 2-naphthol.(26) This means that the rate of excited-state proton transfer by tyrosine to water is on the order of 105s 1. With a fluorescence lifetime near 3 ns for tyrosine, the combined rates for radiative and nonradiative processes approach 109s-1. Thus, the proton transfer reaction is too slow to compete effectively with the other deactivation pathways. 

Given a natural (i.e., no radiationless decay) fluorescence lifetime r0for an isolated fluorophore, one can show that the actual observed lifetime for a real fluorophore near an interface is... 

Orientation, Rotation, and Fluorescence Lifetime of Molecules near Surfaces... 

Both the physics and the chemistry of proximity to a surface can alter the excited-state lifetime and rotational motion of a fluorescent molecule. An extrinsic label attached to BSA has been found to reduce its fluorescence lifetime upon BSA adsorption to fused silica.(95) The decrease is too large to arise from the physical near-field proximity effects discussed in Section 7.3 ... 

W. Lukosz and R. E. Kunz, Fluorescence lifetime of magnetic and electric dipoles near a dielectric surface, Opt. Commun. 20, 195-199 (1977). 

Overview. The proposed near-term research is divided into three areas, which will be pursued simultaneously. The first is the complete characterization of the current mercury-responsive fluorescent chemosensor system, including the measurement of fluorescence lifetimes, to discern the origin of the conformational control of fluorescence. The second is to develop two classes of substituted biaryl acetylenic fluorescent chemosensors, to move the observed fluorescence into the visible region and increase the magnitude of the fluorescence signal, which occurs upon conformational restriction. 

Photochemistry can be used to demonstrate solvent effects in supercritical fluids. The analysis revealed trimodal fluorescence lifetime distributions near the critical temperature, which can be explained by the presence of solvent-solute and solute-solute clustering. This local aggregation causes an increase in nonradiative relaxations and, therefore, a decrease in the observed fluorescence lifetimes. Concentration and density gradients are responsible for these three unique lifetimes (trimodal) in the supercritical fluid, as contrasted with the single lifetime observed in a typical organic solvent. The... 

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