Confocal Microscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 221

Acousto-optic scanning devices are based on the principle that when an acoustic
wave travels in an optical medium, it induces a periodic modulation in the index
of refraction of the material creating a diffraction grating which is responsible for
the deflection of the light beam [ 16 ]. There are two types of acousto-optic (AO)
devices used for beam deflection: AO deflectors and AO modulators. Both of these
devices are very similar and based on the same working principle. An AO deflector
depends on the variation of the acoustic frequency in order to change the angle of
deflection and hence scanning the optical beam. AO modulators are used to change
the amplitude or frequency of the diffracted beam. An advantage over mechanical
scanners is the possibility of direct random access to different points within the
sample. If specific points of interest are identified within the sample, it is possible
to use AO devices to address them individually and sequentially at high speeds.
The frame rates possible with such devices can be very high up to 25 kHz. The
major disadvantage of using AO devices is that they are made of highly dispersive
materials; hence, propagation of ultrashort femtosecond pulses through such media
causes them to disperse and change the pulse shape and duration. This results in low
efficiency of the nonlinearly generated signal [ 17 ].
Faster scanning devices have a physical limit on their speed to achieve higher
image acquisition rates in multiphoton microscopy. An alternative may be to
parallelize the image acquisition process by using more than one focal volume at a
time, thereby reducing the data acquisition time. One of the first schemes proposed
to use the excitation laser more effectively in multiphoton microscopy involved
using a line focus instead of point excitation [ 18 ]. The line focus is created with
cylindrical optics and is scanned in a perpendicular direction relative to the line
with a galvanometric scanner in order to form a 2-D image. Since only one scanner
is involved in this approach, extremely high frame rates are in principle possible,
but the drawback is the loss of optical section property of nonlinear excitation
in the direction of long axis of the beam. A different approach to parallelize the
image acquisition in nonlinear microscopy is to generate an array of focal points.
A combination of lenslet array illumination and delay of a few picoseconds between
individual foci can avoid the interference effects and achieve higher frame rates.
A multifocal multiphoton microscope (MMM) can use the laser power efficiently
and reduce the image acquisition time from 1 s to 10-50 ms [ 19 , 20 ].
The basic geometry for beam scanning is shown in Fig. 6.9 . The basic idea is
to image the laser beam into the back aperture of the microscope objective with
the help of a telecentric lens system. This ensures that the angular displacement
of the scanners is translated into a tilt of the beam in the back aperture without
any beam displacement. This results in a two-dimensional scan in the focal plane
of the objective, as the scanners move. Usually nowadays, an infinity tube-length
objective lens is used together with a tube lens. As shown, the aperture stop, which
limits the numerical aperture of the system, is situated in the back focal plane of
the tube lens and front focal plane of the objective. In order to fill completely the
objective pupil whilst avoiding shading and vignetting problems, the scan mirror
must be placed in the front focal plane of the scan lens. The light from the sample
returns through the optical system and is descanned by the scan mirror. For two-