Fluorescence lifetime studies applications

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. 

In this final section, we summarize the operation and characteristics of the principal vacuum tube and solid state detectors that are available for red/near-IR fluorescence studies. These include conventional photomultipliers, microchannel plate versions, streak cameras, and various types of photodiodes. Detector applicability to both steady-state and time-resolved studies will be considered. However, emphasis will be placed on photon counting capabilities as this provides the ultimate sensitivity in steady-state fluorescence measurements as well as permitting lifetime studies. 

The combination of gain and fast time-response afforded by APDs has found applications in fluorescence lifetime measurements, such devices not being appropriate in steady-state studies. However, these applications have been primarily limited by the extremely small active area of the fastest devices. 

G. Hungerford, The application of spark source fluorescence lifetime spectroscopy to the study of infrared fluorescence, transient species and DCM, Ph.D. thesis. University of Strathclyde, Glasgow, Scotland (1991). 

The application of vb-DMASP to MIPs was continued in subsequent works [64, 65], In these investigations, time-resolved fluorescence spectroscopy was applied to study bulk fluorescent MIP. The imprinted polymer fluorescence quenching with increasing concentrations of aqueous cAMP was determined from the fluorescence lifetime parameters. Two components in the fluorescence decays were identified and assigned to two different types of cavities present in the polymer matrix. One was accessible and open to binding, whereas the other was inaccessible, being buried inside the bulk polymer. The fluorescence lifetime decreased due to the increase in the concentration of the initial target analyte. However, the accessible... 

With a possible resolution of less than 1 ps [65, 66], streak cameras offer the highest temporal resolution of any device currently available. The study of picosecond phenomena is a rapidly expanding field and the state-of-the-art in picosecond techniques was recently reviewed [lc]. Here we are concerned only with the general principles of streak cameras and their application to measuring fluorescence lifetimes. 

Excitation of the porphyrin moiety of 2 in dichloromethane solution yields the first excited singlet state, which can decay according to the pathways detailed in Figure 4. As with P-Q dyad 1, photoinitiated electron transfer competes with other decay processes to yield a C-P -Q charge-separated state. Fluorescence decay studies yielded a fluorescence lifetime r of 0.10 ns for 2 [27]. The hydroquinone form of the triad, 3, in which such electron transfer is not possible, has a fluorescence lifetime of 3.4 ns in the same solvent (see Section 111.A.). Application of Eq. (1) yields an electron transfer rate constant fcj in Figure 4 of 9.7 x 10 s", and consequently a quantum yield for this step of essentially unity. Thus, the addition of the carotenoid moiety to the molecule has had little influence upon the initial photodriven electron transfer step. 

Quenching studies of protein fluorescence provide answers regarding the accessibility of certain internal or external groups to quencher molecules. Another application concerns the study of associative behavior and properties of proteins and membranes. The rationale is that the fluorescence transition is polarized and this polarization can be exploited in time-resolved analysis and interpreted in terms of the rotation or tumbling motion which in turn is determined by the viscosity and structure of the environment of the fluorescing group. In particular, anisotropy decay studies have yielded a great deal of information on the mobility of natural and artificial membranes and/or the dynamics of proteins as well as small molecules in membranes. For such studies fluorescence lifetime labels that can be attached to proteins or that dissolve in membranes have... 

Major Applications Recording materials, biosensors, regulating acidity in gastric cells, for biological silicification studies, 1 fluorescence lifetime characterization of lysosomal pH Safety/Toxicity No data available... 

BODIPY dyes were subjected to modifications in their meso position as the previous fluorescein dyes. Aromatic moieties at this position are perpendicular to the molecular plane but, nevertheless, affect the fluorescence properties of the core [99]. Subtle manipulation of the redox potential can be also read out by changes of the fluorescence lifetime as exemplified in a substrate for phosphoester cleavage [100]. Depending on of the redox properties of the substituents, fluorescence can be turned on and off [101]. When the electrochemical potential of the substituent is altered during a reaction, a fluorogenic probe for this particular reaction is tailored. This was exploited for studying interfacial electron transfer [61]. More applications appear feasible. 

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