Pulse intrinsic fluorescence

Plasma proteins organize on polymer substrates in different ways. Adsorbates are influenced by substrate physicochemical properties and by environmental factors, especially fluid shear and bulk protein distribution. Different types of binding interactions and more than one conformation for adsorbed protein are observed. In the case of albumin, the irreversibly adsorbed conformation, as measured by pulse intrinsic fluorescence, appears to be substantially altered from that of bulk albumin. Microaggregated albumin and undenatured forms are seen at the polymer interface, which are readily desorbed by viscous drag. 

Temporal characteristics at early stages were elucidated by measuring fluorescence intensity with the gate time of 1.74 ns as a function of the delay time. Compared to the laser pulse, the time where the maximum intensity is attained shifts to the early stage as the laser fluence becomes high. Of course, we could not find out any decay component with intrinsic fluorescence lifetime of 17 and 35 ns. It is concluded that an Si - Si annihilation occurs quite efficiently during the pulse width. 

Here, L(ti) is the hei t and At the width of the excitation pulse s 6-fimction component at time t/. F(t) (the intrinsic fluorescence decay) is given by equation 1 and its argument t - t accounts fijr the fiict that the elapsed time varies between when the different subpopulations are created (at times t, ) and when the fluorescence intensity is measured. The (relative) height of L at t( appropriately scales the magnitude of the resulting fluorescence response. The effect of convolving an exponential decay with an excitation pulse of finite width will be seen in Section 2.6.4, where we describe the analysis of pulsed-excitation data. A fuller discussion of the convolution integral may be found in (9). ... 

If intrinsic two-photon absorption cross-sections are the subject of investigation short pulses (ps or fs) should be used and two-photon fluorescence (see below) should be measured to verify the Independence of the TPA process. Most often, two-photon absorption is measured either with the Z-scan technique (see above) or with the nonlinear transmission method [38]. 

At the opposite end of the spectrum, UV sources for CE-LIF are becoming increasingly popular. UV radiation is capable of inducing fluorescence in many intrinsic fluorophores, including a number of biologically relevant molecules. The frequency-doubled Ar-ion laser (257 nm) was one of the first examples reported of UV-excitation for CE-LIF, and yielded improvements in LODs for a number of substances, such as conalbumin (1.4 x 10 M). As another example, a frequency-doubled Kr laser operating at 284 nm has been used for the analysis of neuropeptides and small biomolecules, and exhibited LODs for tryptophan of 800 zmol. In addition to frequency-doubled ion lasers, a number of relatively inexpensive pulsed lasers such as frequency quadrupled YAG (266 nm), KrF (248 nm), and hollow-cathode metal vapor lasers have appeared, which provide deep-UV excitation (e.g., 224 and 248 nm). ... 

In this method the fluorescence decay of the donor after pulse excitation is measured. Therefore the lifetime of the photon emitted upon fluorescence can be measured. This lifetime is the intrinsic property of the donor chromophore and its value is independent of variations in concentration. This fact is a major advantage of time-resolved methods over steady-state methods. If the decay is a single exponential, the measurement of the decay time in the presence and absence of the acceptor is straightforward. That means that, in circumstances where the decay is single exponential and the donor lifetime changes in the presence of the acceptor, then transfer of energy has taken place. The efficiency of this energy transfer can be calculated by ... 

Ideally, the recorded fluorescence decay as function of time, I(t), would correspond to the intrinsic decay if the excitation pulse would be a 8-function. As the... 

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