Biomedical Engineering Reference
In-Depth Information
in the AOM must propagate in the opposite direction with respect to the sum of the two waves in the
AODs.
The RASH microscope is capable of collecting a field of view of 150 × 150 μm
2
with a radial spatial
resolution of ~800 nm. The commutation time between two positions in the focal plane is of the order
of 4 μs. RASH microscopy can be used to record fast physiological events from multiple positions with
near simultaneous sampling. The RASH system AODs can be rapidly scanned between lines drawn in
the membranes of neurons to perform multiplex measurements of the SHG signal monitoring Vm at the
selected locations.
2.2.4.5 Scanning with a Polygonal Mirror
Polygonal mirrors have been utilized in several past commercial and experimental laser scanning
instruments, but are currently not being implemented by the microscope manufacturers. In practice,
the use of polygon mirrors requires considerably more complex optical component design, and the
units are prone to miniscule variations in reflectivity and angle with respect to the axis of rotation.
Known as pyramidal errors, these angular differences produce beam fluctuations that must be opti-
cally corrected. Due to the fact that the number of polygon facets must be proportional to the total
number of raster scan lines, specialized mirrors must often be fabricated in order to build laser scan-
ning instruments. Polygons having 15, 25, or 75 sides must rotate at 63,000, 37,800, or 12,600 revolu-
tions/min, respectively, in order to generate video rate scanning at 15,750 lines/s. These high speeds
require specialized bearings, further complicating instrument design. For these reasons, polygon
mirror-based SHG microscopes are rare and generally relegated to special interest projects (Veilleux
et al., 2008).
2.2.5 Microscope objective Lens
In a laser scanning SHG microscope, the excitation objective determines the spatial resolution of the
microscope. In wide-field microscopy, the spatial resolution achieved with an objective lens is limited
by the diffraction limit of the wavelength used. In a nonlinear laser scanning microscope, the spatial
resolution corresponds to the excitation volume, which is smaller than the diffraction limit (see Figure
2.8a) because of the nonlinear optical properties of the excitation process. Although coherent optical
processes are not in general characterized by a PSF, it is useful to approximate the spatial resolution of
an SHG microscope with the PSF associated with fluorescence excited at the same wavelength. By using
this approximation, we can evaluate the best spatial resolution achievable with an SHG microscope. The
spatial radial (ω
xy
) and axial (ω
z
) resolution for an SHG laser scanning microscope can be approximated
by the following two relationships, respectively (Zipfel et al., 2003b):
0 320
2
.
λ
NA
≤
0 7
.
NA
ω
=
(2.5)
xy
0 325
2
.
λ
NA
>
0 7
.
NA
0 91
.
0 532
2
.
λ
1
2
ω
=
(2.6)
z
n
−
n
−
NA
2
where λ is the excitation wavelength used,
n
is the refractive index of the immersion medium, and NA
is the objective numerical aperture.
