Fluorescence power

Brief exposure to nitrous fumes (up to 3 min) leaves the fluorescent power of the acid-instable fluorescence indicator 254. incorporated into most TLC layers, largely unaffected, so that the nitroaromatics so formed can be detected as dark zones on a green fluorescent background [1]. For purposes of in situ quantitation it is recommended that the fluorescence indicator be destroyed by 10 min exposure to nitrous fumes in order to avoid difficulties in the subsequent evaluation [1]. 

The total fluorescence power collected from the fluorophore distribution by a microscope objective centered in the normal line at a distance r is an integral of f over the objective s aperture which subtends a solid angle fl ... 

Fluorescence is spontaneous radiation that arises because of the stimulation of an atomic or molecular system to energies higher than equilibrium. This is illustrated in Figure 1 for a simple two-level atom. The atom is excited by absorption of a photon of energy hv. If the fluorescence is observed at 90° to a collimated excitation source, then a very small focal volume may be defined resulting in fine spatial resolution. The fluorescence power an optical system will collect is... 

In most fluorescence models where the spectral profile is ignored, the emitted fluorescence power is then simply given as This... 

The total fluorescence power is then obtained using Eqs. 1 and 4 as... 

The modified fluorescence power can be expressed, as in the firee space case, as = 0(T bs inc conveniently be expressed as a... 

Similarly, the total fluorescence power enhancement factor is ... 

A related method has been used to demonstrate that lifetimes as ort as 200 ps can be measured usii the mode noise in a free-running a on-ion laser to produce variations in the excited state population of a fluorophore . Meaairement of the rf power spectmm of the resilting fluctuations then reveals the excited state lifetime. Mode noise contains very high frequency fluctuations which the excited state population cannot follow because of its finite lifetime, and thus these hi frequency components are absent from the rf spectrum of the fluorescence fluctuations. The fluorescence process thus acts like a low pass exponential filter, and comparison of the fluorescence power spectmm with that of the source provides the decay time data, as demonstrated below. 

The quantitative relationship between fluorescence power and analyte concentration can be derived from the number of molecules in the excited state and the radiant power absorbed by the processed sample ... 

The spectrum of lattice-trapped atoms is recorded using a heterodyne technique. Light fluoresced by the trapped atoms is combined with light (frequency shifted by a modulator) from the laser forming the lattice. When the beams mix on a photodiode they create a beat signal at the difference frequency between the fluorescence and the frequency-shifted laser. The power spectrum of the photocurrent is identical to the fluorescence power spectrum, but centered at radio frequency. This heterodyne technique is not sensitive to the frequency jitter of the laser because the jitter is common between the fluorescence and the laser, which acts as a local oscillator. 

Inserting (6.59) with 7l(0 = cpp(0 into (6.58) yields the time-dependent population density Nkit) of the upper level, and therefore also the fluorescence power Ppi(0 = Nk(t)Akm emitted on the transition -> m). The result is... 

Equation (6.61) anticipates a pure exponential decay. This is justified if a single upper level k) is selectively populated. If several levels are simultaneously excited the fluorescence power Rpi(t) represents a superposition of decay functions with different decay times x/t. In such cases the phase shifts < (f2) and the amplitudes a/(l + have to be measured for different modulation frequencies The... 

Fig. 7.2. Laser-induced fluorescence power and population difference AA as a function of laser intensity 7l...

In conventional fluorescence analysis the majority of quantitative measurements is made using fixed wavelengths for excitation and emission. At low concentrations, a plot of the fluorescent power of a solution vi. the concentration of the emitting species ordinarily is linear. As to the sensitivity of fluorescence procedures in analysis, it is convenient to separate the contributions from the properties of the fluorescent molecule itself absolute sensitivity), the performance of the instrument instrumental sensitivity) and the chemistry involved in the preparation of the sample method sensitivity). The absolute sensitivity is determined chiefly by the molar absorptivity and the fluorescence efficiency of the analyte molecule itself. High fluorescence efficiency is usually associated with some rigidity. The method sensitivity takes account of pre-concentration steps in the preparation of the sample on the one hand and the limitations imposed by the fluorescence of the blank on the other. The sensitivities and selectivi-ties attained by fluorescence, phosphorescence and chemiluminescence are hardly paralleled by other techniques. In many cases it is not required that the analyte be isolated from the matrix. 

Figure 3 compares the power collection efficiency of two optical collection schemes, system I, a trans-illumination scheme and system II, an epi-illumination scheme. The power collection efficiency is computed with respect to system 0 gathering the power Pq from a randomly oriented fluorophore located at a distance z > 0 from a glass-water interface using a 1.20NA water immersion objective positioned at z<0. The dotted line describes the fluorescence power Pi collected by system I, consisting of a 1.20NA water... 

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