Biomedical Engineering Reference
In-Depth Information
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FIgurE 2.5 (a) Schematic sketch of a lens-based scanning head for relaying the two scanners. With this configu-
ration, both scanners are placed in a conjugate plane with respect to the objective back-focal plane. (b) Schematic
sketch of a mirror-based scanning head. In this configuration, the mirrors are slightly tilted in order to allow the
positioning of scanners in conjugate planes with respect to the objective back focal plane.
The two spherical mirrors have to be rotated of a small angle along a vertical axis in order to make
space for placing the two galvo-mirrors. In fact, in an ideal geometry, the two galvo-mirrors should
be located exactly in the same point. (The ideal geometry is obtained with the beam orthogonal to the
mirrors surface. This can be obtained only by placing both scanners in the common focus of the two
mirrors.) The rotation of the two spherical mirrors allows to slightly shift in opposite directions the
points in which the galvo-mirrors have to be placed (as in Figure 2.5b where the scanners are displaced
from the common focus of the two mirrors). This optical configuration can be mounted in a set-up with
limited available space but it could introduce a slight astigmatism in the laser beam, affecting the spatial
resolution of the microscope.
2.2.4.4 custom AoDs-Based Scanning Head
The major limiting factor of galvanometric scanners is the scanning time. For this reason, the optical
recording of fast physiological events (of the order of ms) is generally possible only in a single posi-
tion of the field of view by using a line scanning procedure. On the other hand, in principle, the opti-
cal measurement of time-dependent processes at selected locations does not require the production of
images at all. Instead, more time could be spent collecting as many photons as possible from selected
positions, where the image plane intersects the biological objects of interest. Unfortunately, standard
galvo-mirrors require about 1 ms to reach and stabilize a new position; therefore, even implementing a
line scan approach on a selected sample region, the sampling frequency is always below 1 kHz, limiting
the recording of fast physiological processes (such as, e.g., action potentials). On the other hand, high-
speed scanning of a set of points within a plane can be achieved with two orthogonal AODs). In an AOD,
a propagating ultrasonic wave establishes a grating that diffracts a laser beam at a precise angle, which
can be changed within a few microseconds (see Figure 2.6).
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