11 Fluorescent Chemosensors
385
might be related to the excess accumulation of long-lived excited triplet state under
high-power excitation, which accelerates the photo-bleaching.
11.3.4 Two-Photon Absorption Cross Section
Typically, an organic dye absorbs a “single” photon to become excited and then
returns to the ground state via emission (fluorescence), thermal deactivation (internal
conversion and subsequent vibrational relaxation), or intersystem crossing to triplet
state and subsequent thermal deactivation or phosphorescence. However, when using
an extremely intense excitation light, such as femtosecond pulse laser, the dye
may absorb two photons simultaneously. This phenomenon is called “two-photon
absorption” or “two-photon excitation” of the dye (Fig. 11.4a).
After either one-photon or two-photon excitation, the dye molecule decays to
the ground state via photophysical processes. Usually, the wavelength used in twophoton excitation is approximately twice that used in the corresponding single-photon
excitation to maintain the same total excitation energy. This means that two-photon
absorption can occur under significantly red-shifted light compared to single-photon
excitation. As described in Sect. 11.3.1, this gives many advantages in fluorescence
imaging. For instance, two-photon excitation should be very valuable for deep tissue
imaging that requires excitation at long wavelengths (tissue optical window). Moreover, two-photon absorption usually occurs at the focal point of the laser where the
photon flux is extremely high. Therefore, spatially selective excitation of the dye can
be realized.
The efficiency of two-photon absorption by the dye is described by “two-photon
absorption cross section”, which can be estimated by the absolute method (z-scan
method) or the relative method (two-photon excited fluorescence method) (Pawlicki
et al. 2009). The product of two-photon absorption cross section and fluorescence
quantum yield is called the “two-photon action cross section”, which can be regarded
as the theoretical brightness under two-photon excitation.
It should be kept in mind that the wavelength for two-photon excitation is just as
important as the two-photon action cross section. In in vivo imaging, an excitation
wavelength over 900 nm is more preferable than that below 900 nm where several
biomolecules would be excited (Fig. 11.4b, c) (Ahn et al. 2015).
11.3.5 Sensitivity to Environmental Condition
Some dyes vary their fluorescence properties (intensity, color, lifetime, etc.) in
response to environmental conditions (e.g., polarity, viscosity, and pH) (Klymchenko
2017). As might be expected, the utility of such dyes largely depends on their sensitivity (the extent of fluorescence property variations) to the changing in environmental
condition.
385
might be related to the excess accumulation of long-lived excited triplet state under
high-power excitation, which accelerates the photo-bleaching.
11.3.4 Two-Photon Absorption Cross Section
Typically, an organic dye absorbs a “single” photon to become excited and then
returns to the ground state via emission (fluorescence), thermal deactivation (internal
conversion and subsequent vibrational relaxation), or intersystem crossing to triplet
state and subsequent thermal deactivation or phosphorescence. However, when using
an extremely intense excitation light, such as femtosecond pulse laser, the dye
may absorb two photons simultaneously. This phenomenon is called “two-photon
absorption” or “two-photon excitation” of the dye (Fig. 11.4a).
After either one-photon or two-photon excitation, the dye molecule decays to
the ground state via photophysical processes. Usually, the wavelength used in twophoton excitation is approximately twice that used in the corresponding single-photon
excitation to maintain the same total excitation energy. This means that two-photon
absorption can occur under significantly red-shifted light compared to single-photon
excitation. As described in Sect. 11.3.1, this gives many advantages in fluorescence
imaging. For instance, two-photon excitation should be very valuable for deep tissue
imaging that requires excitation at long wavelengths (tissue optical window). Moreover, two-photon absorption usually occurs at the focal point of the laser where the
photon flux is extremely high. Therefore, spatially selective excitation of the dye can
be realized.
The efficiency of two-photon absorption by the dye is described by “two-photon
absorption cross section”, which can be estimated by the absolute method (z-scan
method) or the relative method (two-photon excited fluorescence method) (Pawlicki
et al. 2009). The product of two-photon absorption cross section and fluorescence
quantum yield is called the “two-photon action cross section”, which can be regarded
as the theoretical brightness under two-photon excitation.
It should be kept in mind that the wavelength for two-photon excitation is just as
important as the two-photon action cross section. In in vivo imaging, an excitation
wavelength over 900 nm is more preferable than that below 900 nm where several
biomolecules would be excited (Fig. 11.4b, c) (Ahn et al. 2015).
11.3.5 Sensitivity to Environmental Condition
Some dyes vary their fluorescence properties (intensity, color, lifetime, etc.) in
response to environmental conditions (e.g., polarity, viscosity, and pH) (Klymchenko
2017). As might be expected, the utility of such dyes largely depends on their sensitivity (the extent of fluorescence property variations) to the changing in environmental
condition.