18
H. Miyasaka et al.
molecular motion, where the localization of fluorescence images enables to track the
translational diffusion of individual molecules at very high spatial resolution (typically, 10–30 nm). The diffusional motion of molecules can probe the microscopic
environments of host materials and thus provides the information on the positiondepending properties of host materials. By using this method, we have investigated
the microscopic properties of polymer-based materials, e.g., the evolution of the
property of polymers through cross-linking [26] and photo-curing process [27] and
peculiar diffusion of guest molecules depending on the distance from solid/polymer
interface [28].
Although SMT is a powerful tool, two practical limitations have inhibited the
tracking of large number of molecules with a long measurement time. The first one
is the limited time of tracking due to the photobleaching of fluorescence molecules.
For single-molecule fluorescence imaging with good signal-to-noise (S/N) ratio,
photoexcitation with relatively intense laser is required. This intense excitation
induces the photodegradation of fluorescence molecules typically in several tens
of seconds of the light exposure. The second one is the upper limit of the concentration of guest molecules. For ensuring the single-molecule tracking based on the
localization method, spatial overlap between fluorescence spots of guest molecules
should be avoided during their Brownian motions. Thus, the concentration of guest
molecules should be kept low. Because of these two limitations, it was difficult to
cover the wide region of the specimen by longtime measurements.
The application of the fluorescence OFF–ON switching is one of the methods
to overcome these limitations. In this measurement, most of the molecules in the
specimen are in the fluorescent OFF state, and the number of the molecules in the
fluorescent ON state is kept low to avoid the spatial overlap of fluorescence spots.
The bleaching of ON state by the photodegradation or by the ON → OFF reaction is
recovered by the exposure of the switching light to induce the OFF → ON reaction.
Iteration of the tracking and the OFF → ON light exposure allows the single-molecule
tracking of the large number of molecules for a longtime covering the wide area of
the specimen.
The bleaching of ON state by the photodegradation or by the ON→OFF reaction
is recovered by the exposure of the switching light to induce theOFF→ONreaction.
In this measurement, the OFF → ON reaction is usually induced by the UV light,
which frequently prompts the photodegradation of the dye resulting in the short
measurement time. Accordingly, the new method of the OFF → ON, ON → OFF,
and fluorescence excitation without UV exposure can contribute to the longtime
measurement covering wide region and time-dependent change of the materials. In
the following, we introduce the visible one-color control method for OFF → ON,
ON → OFF, and fluorescence excitation, which was developed in this project [29].
Figure 1.18a shows the structures of the open- and closed-ring isomers fluorescent diarylethene (fDAE). The quantum yield of the cyclization (fluorescence
OFF → ON) reaction in dioxane solution is 0.23, while that of the cycloreversion
(fluorescence ON → OFF) is <10
−5 . As Fig. 1.18b shows, the absorption band of
the open-ring isomer locates in the region shorter than 450 nm, while the closed-ring
H. Miyasaka et al.
molecular motion, where the localization of fluorescence images enables to track the
translational diffusion of individual molecules at very high spatial resolution (typically, 10–30 nm). The diffusional motion of molecules can probe the microscopic
environments of host materials and thus provides the information on the positiondepending properties of host materials. By using this method, we have investigated
the microscopic properties of polymer-based materials, e.g., the evolution of the
property of polymers through cross-linking [26] and photo-curing process [27] and
peculiar diffusion of guest molecules depending on the distance from solid/polymer
interface [28].
Although SMT is a powerful tool, two practical limitations have inhibited the
tracking of large number of molecules with a long measurement time. The first one
is the limited time of tracking due to the photobleaching of fluorescence molecules.
For single-molecule fluorescence imaging with good signal-to-noise (S/N) ratio,
photoexcitation with relatively intense laser is required. This intense excitation
induces the photodegradation of fluorescence molecules typically in several tens
of seconds of the light exposure. The second one is the upper limit of the concentration of guest molecules. For ensuring the single-molecule tracking based on the
localization method, spatial overlap between fluorescence spots of guest molecules
should be avoided during their Brownian motions. Thus, the concentration of guest
molecules should be kept low. Because of these two limitations, it was difficult to
cover the wide region of the specimen by longtime measurements.
The application of the fluorescence OFF–ON switching is one of the methods
to overcome these limitations. In this measurement, most of the molecules in the
specimen are in the fluorescent OFF state, and the number of the molecules in the
fluorescent ON state is kept low to avoid the spatial overlap of fluorescence spots.
The bleaching of ON state by the photodegradation or by the ON → OFF reaction is
recovered by the exposure of the switching light to induce the OFF → ON reaction.
Iteration of the tracking and the OFF → ON light exposure allows the single-molecule
tracking of the large number of molecules for a longtime covering the wide area of
the specimen.
The bleaching of ON state by the photodegradation or by the ON→OFF reaction
is recovered by the exposure of the switching light to induce theOFF→ONreaction.
In this measurement, the OFF → ON reaction is usually induced by the UV light,
which frequently prompts the photodegradation of the dye resulting in the short
measurement time. Accordingly, the new method of the OFF → ON, ON → OFF,
and fluorescence excitation without UV exposure can contribute to the longtime
measurement covering wide region and time-dependent change of the materials. In
the following, we introduce the visible one-color control method for OFF → ON,
ON → OFF, and fluorescence excitation, which was developed in this project [29].
Figure 1.18a shows the structures of the open- and closed-ring isomers fluorescent diarylethene (fDAE). The quantum yield of the cyclization (fluorescence
OFF → ON) reaction in dioxane solution is 0.23, while that of the cycloreversion
(fluorescence ON → OFF) is <10
−5 . As Fig. 1.18b shows, the absorption band of
the open-ring isomer locates in the region shorter than 450 nm, while the closed-ring