648
S. P. Singh and S. Siddhanta
has been used to study the intracellular dynamics of a protein ‘paxillin’. This protein
is known to be associated with regulating cellular adhesion and turnover [35]. Using
FCS authors measured the dynamics of paxillin in the cytoplasm focusing primarily
on its binding and unbinding capacities [35]. Overall, their findings data reveal rapid
binding dynamics of paxillin in newly forming and maturing regions of adhesions
and high affinity and clustering in more mature or disassembling adhesions.
4.3 Two-Photon Excitation Fluorescence Microscopy
(TPEFM)
Wolfgang Kaiser for the first time demonstrated the experimental feasibility of
TPEFM [36]. Some of the major advantages of TPEFM over other live-cell imaging
techniques include higher penetration up to 1 mm, minimal phototoxicity, and
reduced scattering losses [37]. In contrast to routinely employed fluorescence
imaging, the excitation wavelengths in two-photon microscopy are often in the near
or mid-infrared range [38]. Similar to confocal fluorescence imaging, this method
also depends upon the absorption of photons for fluorophore excitation. The only
difference is that two or three photons are required to be absorbed within a few
femtoseconds to produce signals. To improve the event of two photons interacting
with the fluorophore together often high-intensity lasers are used for excitation in the
focal area. The raster scanning of the laser light generates an intensity profile across
the whole area of acquisition. As all the emitted light originates from a single focal
spot, the background noise is significantly reduced [37, 39]. Due to the low energy of
the excitation beam, the damage to the samples is minimum, therefore this method is
highly recommended to study live specimens. Often a titanium sapphire laser emitting pulsed near-infrared beam is used. Before focusing it on the desired spot, the
beam passes through a pair of galvo mirrors, dichroic filters. Finally, the emitted fluorescence is collected through photomultiplier tubes. The biomedical applications of
TPEFM range from cancer diagnosis, neuronal imaging, and monitoring embryonic
development [40–42]. Nervous system imaging is one of the most common applications of two-photon imaging [43]. Another major application of this method is in
following maturation and migration of immune cells. Benninger et al. has reviewed
all the major applications of two-photon microscopy [44]. In Fig. 5 authors for the first
time imaged membrane lipid order in a whole, living vertebrate organism (zebrafish)
with help of a phase-sensitive, membrane-partitioning probe Laurdan in conjunction with multiphoton microscopy. They also quantitatively analyzed the membrane
order, which showed high lipid order in the apical surfaces of polarized epithelial
cells [45].
S. P. Singh and S. Siddhanta
has been used to study the intracellular dynamics of a protein ‘paxillin’. This protein
is known to be associated with regulating cellular adhesion and turnover [35]. Using
FCS authors measured the dynamics of paxillin in the cytoplasm focusing primarily
on its binding and unbinding capacities [35]. Overall, their findings data reveal rapid
binding dynamics of paxillin in newly forming and maturing regions of adhesions
and high affinity and clustering in more mature or disassembling adhesions.
4.3 Two-Photon Excitation Fluorescence Microscopy
(TPEFM)
Wolfgang Kaiser for the first time demonstrated the experimental feasibility of
TPEFM [36]. Some of the major advantages of TPEFM over other live-cell imaging
techniques include higher penetration up to 1 mm, minimal phototoxicity, and
reduced scattering losses [37]. In contrast to routinely employed fluorescence
imaging, the excitation wavelengths in two-photon microscopy are often in the near
or mid-infrared range [38]. Similar to confocal fluorescence imaging, this method
also depends upon the absorption of photons for fluorophore excitation. The only
difference is that two or three photons are required to be absorbed within a few
femtoseconds to produce signals. To improve the event of two photons interacting
with the fluorophore together often high-intensity lasers are used for excitation in the
focal area. The raster scanning of the laser light generates an intensity profile across
the whole area of acquisition. As all the emitted light originates from a single focal
spot, the background noise is significantly reduced [37, 39]. Due to the low energy of
the excitation beam, the damage to the samples is minimum, therefore this method is
highly recommended to study live specimens. Often a titanium sapphire laser emitting pulsed near-infrared beam is used. Before focusing it on the desired spot, the
beam passes through a pair of galvo mirrors, dichroic filters. Finally, the emitted fluorescence is collected through photomultiplier tubes. The biomedical applications of
TPEFM range from cancer diagnosis, neuronal imaging, and monitoring embryonic
development [40–42]. Nervous system imaging is one of the most common applications of two-photon imaging [43]. Another major application of this method is in
following maturation and migration of immune cells. Benninger et al. has reviewed
all the major applications of two-photon microscopy [44]. In Fig. 5 authors for the first
time imaged membrane lipid order in a whole, living vertebrate organism (zebrafish)
with help of a phase-sensitive, membrane-partitioning probe Laurdan in conjunction with multiphoton microscopy. They also quantitatively analyzed the membrane
order, which showed high lipid order in the apical surfaces of polarized epithelial
cells [45].