Biomedical Engineering Reference
In-Depth Information
and components, including optics, electronics, detectors, light sources, etc., has
greatly expanded fluorescence microscopy imaging functionalities and enabled
the development of many sophisticated fluorescence microscopy techniques (see
Sect.
3.3
). Moreover, computer-controlled fluorescence microscopes equipped with
sophisticated accessories and software have facilitated the imaging of live speci-
mens under physiological conditions as well as expended the variety of experimental
manipulations [
8
]. For example, motorized fluorescence microscopes equipped with
devices capable of maintaining temperature, CO
2
, humidity, and focus provide
biologists with the capability to monitor subcellular events over a few hours to days
[
9
-
11
]; the usage of optical tweezers on a motorized fluorescence microscope offers
biologists the ability to manipulate subcellular structures or particles [
12
].
Fluorescence microscopy has become an essential tool in the life sciences due
to attributes not as readily available with other optical microscopy techniques. The
general advantages of fluorescence microscopy techniques include the high degree
of specificity amidst nonfluorescing material, the exquisite sensitivity which is
able to detect individual fluorescent molecules, and the high temporal and spatial
(available in 3-D) resolution. In this chapter, basics of fluorescence and fluores-
cent probes are introduced (Sect.
3.2
), and several commonly used and advanced
fluorescence microscopy imaging techniques are briefly presented (Sect.
3.3
). This
chapter focuses on fluorescence lifetime imaging microscopy (FLIM) and describes
the design and calibration of a two-photon excitation time-correlated single-photon
counting (TPE-TCSPC) FLIM system built in our laboratory (Sect.
3.4
). The
combination of TPE-TCSPC FLIM system and Forster resonance energy transfer
(FRET) helped to investigate protein-protein interactions in living specimens. In this
chapter, we described the development of TPE-TCSPC FLIM system to investigate
the homodimerization of the transcription factor CCAAT/enhancer-binding protein
alpha (C=EBP˛) in live mouse pituitary cell nuclei (Sect.
3.4
).
3.2
Basics of Fluorescence and Fluorescent Probes
Fluorescence is one of the many different luminescence processes and is the
emission of light from the excited singlet state of a substance that has absorbed
light or other electromagnetic radiation of a different wavelength. The excitation of
molecules by light occurs via the interaction of molecular dipole transition moments
with the electric field of the light and, to a much lesser extent, interaction with the
magnetic field. The fluorescence processes following light absorption and emission
are usually illustrated by the Jablonski diagram (see Fig.
3.1
), and the energy of the
emission is typically less than that of absorption. Thus, the fluorescence emission of
a fluorophore is usually red shifted (longer wavelength) compared to its excitation -
a phenomenon called the Stokes shift (see Chapter
1
in [
13
]). Besides the absorption
and emission spectra, the important photophysical parameters of a fluorophore
include (1) the fluorescence lifetime (described in Sect.
3.4
), (2) the quantum yield
(the number of emitted photons relative to the number of absorbed photons) which
represents the fluorescence emission efficiency of the fluorophore, and (3) the molar
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