Fluorescence Lifetime of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 80

other extreme are streak cameras having the highest time resolution in this kind of
experiments, even down to 1 ps. Here, photoelectrons are deflected by a rapidly

changing electric field and then reconverted into light by a luminescence screen.
A two-dimensional image of the luminescence then provides time resolution in the
dimension along the electric field, i.e., the time axis. Often, the second axis is used
for spectral information.
Phase fluorimetry is a completely different method, which works without pulsed
excitation (Fig. 5b ). Here, the intensity of the excitation light is modulated in time
with a sine wave. The fluorescence arises with some retard, i.e., a phase-shift

<

D f,
but it obeys the same modulation frequency o. However, as the spontaneous
fluorescence is a stochastic process of uncorrelated emitters, also the demodulation
m of the fluorescence light drops as the inverse of the demodulation frequency, i.e.,
2po 1 , approaches t Fl . Thus, both D f and m can be used for the determination of
t Fl . In an ideal case, both values coincide [ 47 - 49 ].

2.2 Fluorescence Lifetime Imaging Microscopy

In principle, most of the aforementioned experiments can be used in imaging by
performing lifetime experiments in a diffraction-limited spot. Consecutive record-
ing of t Fl at neighbouring points in one dimension and then a line-by-line scan
yields a two-dimensional representation of t FL , which can be extended to the third
spatial dimension in confocal and multiphoton microscopy. Such experiments were
performed with time-correlated single-photon counting, frequency modulation and
even with streak-cameras; pump-probe experiments were hardly performed yet. As
the recording of t fl at each pixel needs at least a fraction of an ms, a whole image is
not acquired faster than several seconds. Faster processes cannot be visualized by
this technique.
On the contrary, there are imaging experiments where the fluorescence is
collected from a large area. The lifetime information is provided by some electron-
ics, mostly an image intensifier, in front of a charge-coupled device (CCD). The
fluorescence intensity in dependence of an adjustable time-gate, which is applied to
the high-voltage of the image intensifier, enables extraction of t Fl from at least two
images. Alternatively, modulation of the sensitivity of the detector array allows the
combination of phase fluorimetry with microscopy.

2.3 Lifetime Differences Between Microscopic and Cuvette
Experiments

Although t Fl measured in cuvette experiments should be identical to the values
obtained in microscopy, several reasons can be identified why deviations are