Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 93

are necessary for intensity-based measurements of sensitized emission from the
acceptor (see Sect. 3.3.4 ). The fluorescence lifetime is not sensitive to the change
in fluorophore concentration, excitation light intensity, or light scattering; these
facts make FLIM-FRET an accurate method for FRET measurements. A number
of FLIM-FRET methods have been developed for studying protein-protein inter-
actions and investigation of signaling events in a variety of biological systems
[ 142 , 163 , 164 ]. For example, we developed a fast widefield TD FLIM system using
a gated intensifier camera and TPE [ 152 ] and also implemented a TPE-TCSPC
FLIM system (described below). The protocols for applying both TD and FD FLIM
techniques to FRET studies are available in the literature [ 165 , 166 ].
Other than measuring FRET, FLIM has also been applied to in many other
investigations, as seen from the following few examples. High-speed widefield
FLIM was employed to measure the change in calcium concentration in live
cells using calcium-sensitive probes (e.g., Oregon Green) [ 144 , 167 ]. TPE-TCSPC
FLIM was used to map the microenvironment (response of rigor cross-bridges to
stretch) of the myosin essential light chain in skeletal muscle fibers by probing
the microenvironment of the interface region of the myosin lever arm domain with
coumarin [ 168 ]; FLIM was applied to characterize dental disease through imaging
endogenous fluorophores in dental tissues [ 169 ]. Multiphoton FLIM tomography
(3-D + lifetime) distinguished between different types of endogenous fluorophores
in human skin [ 170 ], and multiphoton multispectral FLIM has shown the potential
to become a valuable technique in stem cell research [ 171 ]. Nicotinamide adenine
dinucleotide (NAD C ) is a coenzyme found in all living cells and carries electrons
from one reaction to another through redox reactions in metabolism. When NAD C
accepts electrons from other molecules, it forms NADH which is highly fluorescent
with peak absorption and emission maxima at 340 and 460 nm (see Chapter 3 in
[ 13 ]) and can be imaged with TPE microscopy [ 172 ]. Thus, NADH can serve
as a convenient noninvasive fluorescent probe of the metabolic state. Since the
fluorescence lifetimes of NADH usually increase upon its binding to proteins, FLIM
has been applied to detect the free (shorter lifetime) and bound (longer lifetime)
forms of NADH, showing promise in cancer research [ 173 - 176 ].

3.4.2 Design of the TPE-TCSPC FLIM System

TCSPC is a commonly used time-domain FLIM method, and the basic principle
of TCSPC is described in the literature [ 147 , 177 ]. The key of TCSPC FLIM is
to be able to record both the spatial (x, y, z ) information of each detected photon
and its arrival time relative to the corresponding excitation event. In general, the
major components of a TCSPC FLIM system include a microscope, a pulsed laser
(one- or two-photon), a high-sensitivity detector (PMT or APD), a computer plus
TCSPC devices, and a photodiode that generates the laser pulse reference fed to the
TCSPC devices. The repetition rate and the pulse width of the pulsed laser should
be determined depending on the lifetime values to be measured. For example, the
measurement of nanosecond lifetimes requires megahertz repetition rates.