384
Y. Niko and S. Watanabe
in a given time. On the other hand, the brightness estimated from microscopy is
apparently more reliable, therefore this method is recommended for bioimaging.
In any case, it should be noted that the illumination power also tends to affect the
brightness of the dyes (Reisch and Klymchenko 2016).
An important consideration comes from the above discussion: between two dyes
with the same theoretical brightness, the one exhibiting higher fluorescence quantum
yield might be more useful than the other, because it is less likely to generate nonfluorescent species during laser excitation.
11.3.3 Photostability
The main drawback of organic dyes is that they are easily photo-bleached compared
to inorganic dyes. This often becomes an obstacle in bioimaging, which usually
uses a high-power laser as the excitation source. Several mechanisms have been
proposed for photo-bleaching. The main one is the generation of excited triplet
state of the dye via intersystem crossing after photo-excitation, which results in
photochemical reaction like the Norrish type (IUPAC 1997a, b). In addition, dye
in the triplet excited state may transfer its excitation energy to molecular oxygen,
thereby generating highly reactive singlet oxygen that can attack several kinds of
molecules including the dye itself (Zheng et al. 2014). Another mechanism is photoisomerization, typified by the isomerization between trans-stilbene (fluorescent) and
cis-stilbene (non-fluorescent) (Görner and Kuhn 2007).
Photostable dyes have the advantage of enabling long-term imaging. Moreover,
dyes with high photostability are indispensable for some techniques such as singlemolecule tracking (Zheng et al. 2014) and super resolution imaging (e.g., stimulated
emission depletion microscopy (STED)) (Wang et al. 2017). In addition, the photostability would be important for storing the dye. On the other hand, extreme photostability is not always useful in fluorescence imaging, because in one technique the
dye is intentionally bleached.
Photostability of the dye is often characterized by a spectrofluorometer equipped
with xenon lamp. In this method, the dye is continuously excited for a given time
while the decay of fluorescence intensity is monitored. Photostability of the dye can
be characterized by the decay constant, which is easily estimated from the analysis
of the fluorescence decay curve, by assuming that photodegradation of the excited
dye is a first-order reaction. This method is applicable when laser is the excitation
source. Alternatively, photostability of the dye can be estimated using a microscope
equipped with laser, by counting the total number of photons emitted by the dye
until photo-bleaching (see Table 11.1). It is known that small organic dyes generally deliver between 10
5 and 10
6 photons for periods of 10–100 s under the strong
illumination required for their imaging (≈1 kW cm
−2 ). Note that photostability of
the dye obtained from spectrofluorometer and microscopy are not always identical,
like the case of brightness characterization (Reisch and Klymchenko 2016). This
Y. Niko and S. Watanabe
in a given time. On the other hand, the brightness estimated from microscopy is
apparently more reliable, therefore this method is recommended for bioimaging.
In any case, it should be noted that the illumination power also tends to affect the
brightness of the dyes (Reisch and Klymchenko 2016).
An important consideration comes from the above discussion: between two dyes
with the same theoretical brightness, the one exhibiting higher fluorescence quantum
yield might be more useful than the other, because it is less likely to generate nonfluorescent species during laser excitation.
11.3.3 Photostability
The main drawback of organic dyes is that they are easily photo-bleached compared
to inorganic dyes. This often becomes an obstacle in bioimaging, which usually
uses a high-power laser as the excitation source. Several mechanisms have been
proposed for photo-bleaching. The main one is the generation of excited triplet
state of the dye via intersystem crossing after photo-excitation, which results in
photochemical reaction like the Norrish type (IUPAC 1997a, b). In addition, dye
in the triplet excited state may transfer its excitation energy to molecular oxygen,
thereby generating highly reactive singlet oxygen that can attack several kinds of
molecules including the dye itself (Zheng et al. 2014). Another mechanism is photoisomerization, typified by the isomerization between trans-stilbene (fluorescent) and
cis-stilbene (non-fluorescent) (Görner and Kuhn 2007).
Photostable dyes have the advantage of enabling long-term imaging. Moreover,
dyes with high photostability are indispensable for some techniques such as singlemolecule tracking (Zheng et al. 2014) and super resolution imaging (e.g., stimulated
emission depletion microscopy (STED)) (Wang et al. 2017). In addition, the photostability would be important for storing the dye. On the other hand, extreme photostability is not always useful in fluorescence imaging, because in one technique the
dye is intentionally bleached.
Photostability of the dye is often characterized by a spectrofluorometer equipped
with xenon lamp. In this method, the dye is continuously excited for a given time
while the decay of fluorescence intensity is monitored. Photostability of the dye can
be characterized by the decay constant, which is easily estimated from the analysis
of the fluorescence decay curve, by assuming that photodegradation of the excited
dye is a first-order reaction. This method is applicable when laser is the excitation
source. Alternatively, photostability of the dye can be estimated using a microscope
equipped with laser, by counting the total number of photons emitted by the dye
until photo-bleaching (see Table 11.1). It is known that small organic dyes generally deliver between 10
5 and 10
6 photons for periods of 10–100 s under the strong
illumination required for their imaging (≈1 kW cm
−2 ). Note that photostability of
the dye obtained from spectrofluorometer and microscopy are not always identical,
like the case of brightness characterization (Reisch and Klymchenko 2016). This