by the evanescent-wave illumination is far less than those excited by the epifluorescence illumination. This and other types of near-field illumination have
been proposed [16]. This feature was later combined with high-sensitivity imaging
technique to visualize behavior of fluorescently labeled ATP molecules on the
myosin molecule attached to glass surface [17]. Figure 3.22c shows an example of
the implementation of the evanescent excitation in an inverted epi-fluorescence
microscope. The laser beam used for the evanescent excitation is introduced into
the coverslip by an optically coupled rectangular prism. The multiple total internal
reflections of the laser beam creates evanescent wave at many locations. The cell in
the spot can be visualized both in the epifluorescence and evanescent-wave excited
fluorescence modes. The left panel of Fig. 3.22d shows an epi-fluorescence image of
a cell in which actin filaments were labeled with a fluorophore, rhodamine. A
number of actin filaments run within the whole cell body and peripheral membranous protrusion, lamellipodia (black and white arrows). On the other hand, under the
evanescent-wave illumination (the right panel of Fig. 3.22d), actin filaments (eg.,
white arrow pointing as the same filament as in the left panel) in lamellipodia are
visible, whereas those in the cell body are visible to a much less extent. This is
interpreted as that in the central region of the cell the filaments reside far from the
interface, where the evanescent illumination is weak due to the decay. Thus, with the
evanescent-wave excitation one can estimate how far the intracellular structure is
located from the glass-water interface. A drawback, as compared with the laser
scanning confocal microscopy, is the difficulty in the quantitation of the separation.
However, in some cases, the separation of the distance between the bottom surface of
the cell and the glass surface has been successfully estimated [18, 19]. In these cases,
the geometrically simple configuration of the system (eg., the cell adhered to a flat
surface) enabled the quantitative estimation of the separation. This method has been
also applied to observe the process of exocytosis [20]. Another feature of the
evanescent wave is that the polarization of the excitation is uniquely defined. With
this feature one can obtain the information about the orientation of the emission
dipole of the fluorophore near or at the interface [21, 22].
⁄
ä
Fig. 3.22 (continued) at the position of the cell illuminate the ventral (lower) surface of the cell. The
emitted fluorescence is collected by the objective (either water immersion or air) and received by the
cooled CCD camera. The acquired image is stored on the computer for later analysis. The same
system is also used to perform the epi-fluorescence microscopy. Panel D, comparison of the
epi-fluorescence image and the evanescent-wave exited fluorescence image of a cell labeled for
actin filaments with rhodamine. The Nd-YAG laser (wavelength ¼ 532 nm) and a mercury-arc lamp
(wavelength was selected by an optical filter to be 546 nm) were used as light sources. As described
in the text, actin filaments in the cell body are visible by the epi-fluorescence mode (black arrow in
the left panel), while the peripheral actin filament bundles (white arrows in both panels) are visible
by the evanescent-wave excitation (images taken by Yosuke Senju, Department of Physics, Tohoku
University)
3.7 Fluorescence Method
53
been proposed [16]. This feature was later combined with high-sensitivity imaging
technique to visualize behavior of fluorescently labeled ATP molecules on the
myosin molecule attached to glass surface [17]. Figure 3.22c shows an example of
the implementation of the evanescent excitation in an inverted epi-fluorescence
microscope. The laser beam used for the evanescent excitation is introduced into
the coverslip by an optically coupled rectangular prism. The multiple total internal
reflections of the laser beam creates evanescent wave at many locations. The cell in
the spot can be visualized both in the epifluorescence and evanescent-wave excited
fluorescence modes. The left panel of Fig. 3.22d shows an epi-fluorescence image of
a cell in which actin filaments were labeled with a fluorophore, rhodamine. A
number of actin filaments run within the whole cell body and peripheral membranous protrusion, lamellipodia (black and white arrows). On the other hand, under the
evanescent-wave illumination (the right panel of Fig. 3.22d), actin filaments (eg.,
white arrow pointing as the same filament as in the left panel) in lamellipodia are
visible, whereas those in the cell body are visible to a much less extent. This is
interpreted as that in the central region of the cell the filaments reside far from the
interface, where the evanescent illumination is weak due to the decay. Thus, with the
evanescent-wave excitation one can estimate how far the intracellular structure is
located from the glass-water interface. A drawback, as compared with the laser
scanning confocal microscopy, is the difficulty in the quantitation of the separation.
However, in some cases, the separation of the distance between the bottom surface of
the cell and the glass surface has been successfully estimated [18, 19]. In these cases,
the geometrically simple configuration of the system (eg., the cell adhered to a flat
surface) enabled the quantitative estimation of the separation. This method has been
also applied to observe the process of exocytosis [20]. Another feature of the
evanescent wave is that the polarization of the excitation is uniquely defined. With
this feature one can obtain the information about the orientation of the emission
dipole of the fluorophore near or at the interface [21, 22].
⁄
ä
Fig. 3.22 (continued) at the position of the cell illuminate the ventral (lower) surface of the cell. The
emitted fluorescence is collected by the objective (either water immersion or air) and received by the
cooled CCD camera. The acquired image is stored on the computer for later analysis. The same
system is also used to perform the epi-fluorescence microscopy. Panel D, comparison of the
epi-fluorescence image and the evanescent-wave exited fluorescence image of a cell labeled for
actin filaments with rhodamine. The Nd-YAG laser (wavelength ¼ 532 nm) and a mercury-arc lamp
(wavelength was selected by an optical filter to be 546 nm) were used as light sources. As described
in the text, actin filaments in the cell body are visible by the epi-fluorescence mode (black arrow in
the left panel), while the peripheral actin filament bundles (white arrows in both panels) are visible
by the evanescent-wave excitation (images taken by Yosuke Senju, Department of Physics, Tohoku
University)
3.7 Fluorescence Method
53