Biomedical Engineering Reference
In-Depth Information
An alternative solution with a lower cost is represented by femtosecond-pulsed fiber lasers. These
compact and robust lasers provide a pulsed emission comparable with that of the Ti:sapphire in terms of
pulse width and repetition rate, but they cannot be tuned. In general, they are preferably used in appli-
cations in which tunability is not required. Their cost is in the order of 20 kEuro and their maximum
power is in the order of W.
Although high repetition rate sources (50-100 MHz) are most commonly used for SHG microscopy,
amplified systems with a repetition rate in the range of few kHz and pulse width in the range of tens of
fs can also be used (Theer et al., 2003, Qiao et al., 2008). These systems have the advantage of provid-
ing shorter pulses with corresponding wider spectral range and hence the capability to excite multiple
fluorophores at the same time. On the other hand, since potential phototoxic effects depend on the 4th-
5th or higher power of the excitation intensity peak, while the signal depends on the 2nd power, these
systems can cause stronger unwanted side effect with respect to a corresponding high repetition rate
source providing the same average light power.
2.2.3 Laser Power Adjustment
Femtosecond-pulsed laser sources used for SHG microscopy commonly provide a power output
higher than that required for safely imaging biological samples and very often they do not allow a
fine regulation of the emitted mean power. For this reason, a proper attenuation system is required
for regulating the amount of optical power used for imaging. Laser power can be adjusted by using
a neutral density filter ring with variable optical density: the filter can be rotated manually or with
a motorized rotational mount allowing power remote control. The use of such a system could have
problems with extremely high-power sources damaging the filter coating and it offers a limited
dynamic range (most common filters have an optical density varying between 0.1 and 2, and hence
a dynamic range of about 0.01-0.9). An alternative is offered by polarization optics. Laser output is
very often polarized so that a half-wave-plate can be used in combination with a polarization beam
splitter cube to adjust the laser power. The optical power transmitted through the polarization beam
splitter cube depends on the rotation angle of the wave plate. As for the neutral density filter of the
above, the wave plate can be rotated manually or with a motorized rotational mount to be controlled
in remote. All these optical elements have to be chosen for sustaining the high intensity of femtosec-
ond pulses with particular attention to the beam splitter cube. For this purpose, Glan-Laser or Glan-
Taylor beam splitter cubes are suggested because they can be used with high peak power and have
a higher extinction ratio for the unwanted polarization, allowing to obtain an extended dynamic
range of about 0.001-1.
2.2.4 Scanning Head
The scanning head is one of the most important parts of an SHG microscope. It allows to rapidly scan
the laser beam focus across the specimen under investigation. Laser beam tilting required for scanning
is generally achieved by using galvanometric mirrors (galvo-mirrors), acousto-optic deflectors (AODs)
or polygonal rotating mirrors. In this section, after having introduced the most popular scanners used
in SHG microscopy, we describe four alternative solutions to be adopted for the scanning head: one
commercial, and the other three custom-made based on galvo-mirrors, AODs, or polygonal mirrors.
2.2.4.1 Scanners
Laser scanning microscopy has proven to be a useful tool for examining cells and tissues. However,
many interesting biological processes occurring on the millisecond timescale cannot be revealed by
laser scanning microscopy because the imaging speed is limited by the bandwidth of the scanners used.
Various approaches can be adopted in order to overcome this limitation and have a faster scanning
system.
