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By Keith J.

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Sample text

T (Fig. 2-5, right). In the frequency-domain method, the sample is excited with intensity-modulated light (Fig. 2-6). The frequency of light intensity modulation is comparable to the reciprocal of the decay time T, which is much smaller than the frequency of the electric component of an electromagnetic wave of the light. Because of the lifetime of the fluorophore, the emission is delayed in time and modulation decreases relative to the excitation. This delay, measured as a phase shift (cp) and demodulation (m), can be used to calculate the decay time.

We want to stress that for a perfectly labeled system, Co = CAHowever, donor or acceptor fractional labeling (underlabeling) is often a concern when dealing with biological systems. In the presence of FRET, part of the excitation energy of the donor will be transferred to the acceptor. 10 ' 49 -r 1 1 1 1 /» / \ > . 02 1 \ \ \ \ \ \ / / nnn 1 "" 400 T 4 -I I... N 15 E Lo 1 / 1 \ 1 \ ' LU •D 1 /\ \ \ \ ^ ^'^ * \. 1 J 1 1 '^N >v \ J ""^ J \ 500 . , • " 1 550 1 1 1 600 1 650 T-^*-agj 700 Wavelength [nm] FIGURE 2-15 Absorption and normalized emission spectra for fluorescein and rhodamine in water.

The fluorescence spectrum of the donor is directly proportional to transfer efficiency, decreasing as transfer efficiency increases. The emission of the acceptor also depends on transfer efficiency. However, it is weighted by the ratio of extinction coefficients at a given excitation wavelength and relative concentrations of donor and acceptor. This feature can be an important complication when one is dealing with an imperfectly labeled system, which is frequently the case for biological systems.

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