Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 92

of a TPE-TCSCP FLIM instrument is described in detail in Sect. 3.4.2 . PMT arrays
have also been employed in TCSPC FLIM systems to obtain spectrally resolved
lifetimes [ 149 , 150 ]. To achieve faster lifetime imaging speeds, the camera-based
TD FLIM techniques have been developed using high-speed gated image intensifier
cameras [ 151 - 154 ] or streak cameras [ 155 , 156 ]. A gated image intensifier camera
can be operated at a superfast speed to detect photons within a time (gating) window
for a few hundred picoseconds to a few milliseconds relative to the excitation pulse
[ 152 ]. In gating-camera FLIM, a number of images are acquired in sequential gating
windows after the excitation pulse to estimate the lifetimes, and extracting single-
component lifetimes requires collecting two gated images at minimal, which only
takes a few seconds [ 152 , 153 ]. A streak camera can be operated to transform the
temporal profile of a light pulse into a spatial profile on a detector by causing a
time-varying deflection of the light across the width of the detector [ 155 ]. In streak-
camera FLIM, a number (y-dimension) of 2-D (x-dimension, time relative to the
excitation pulse) images are acquired to estimate an XY 2-D lifetime distribution,
and each line of an image consists of the time-resolved information for a pixel
location [ 155 ].
The FD method uses a modulated light source to excite a fluorophore and
measures the phase shift(s) and amplitude attenuation(s) of the emission signal
relative to the excitation source, which are then analyzed to estimate the fluores-
cence lifetime. The fundamental modulation frequency of the excitation source is
chosen depending upon the fluorescence lifetime to be measured - e.g., megahertz
should be used for measuring nanosecond (ns) fluorescence lifetimes. The phase
shift(s) and amplitude attenuation(s) can be measured by a detector modulated at
the same frequency as the excitation source (homodyne methods) or a frequency
slightly different (a few hundred to a few thousand hertz) from the excitation
source (heterodyne methods). Similar to the TD FLIM techniques, a number of
FD FLIM techniques have also been developed and implemented on both single-
photon or multiphoton scanning [ 146 ] and camera-based spinning-disk or widefield
microscopes [ 157 - 160 ]. Spectrally resolved FD FLIM system is also available
[ 161 ]. In addition, a recently developed FD FLIM technique called digital FD FLIM
employs a pulsed excitation source and does not modulate the detector at all, thus
allowing for the acquisition of the entire fluorescence signal [ 162 ].
One of the major FLIM applications is to measure FRET (see Sect. 3.3.4 )
between fluorescent molecules, although the acceptor molecules need not be
fluorescent. Using FLIM, FRET events can be identified by measuring the reduction
in the donor lifetime that results from quenching in the presence of an acceptor,
and the energy transfer efficiency (E) can be estimated from the donor lifetimes
determined in the absence ( D - unquenched lifetime) and the presence ( DA -
quenched lifetime) of the acceptor (Eq. 3.3 ) (see Chapter 13 in [ 13 ]).

DA
D

E D 1 (3.3)

Since only donor signals are measured for determining E in FLIM-FRET, the
method does not usually require the corrections for spectral bleedthrough that