Biomedical Engineering Reference
In-Depth Information
According to these equations, a beam focus can be scanned across the focal plane by tilting the laser
beam with respect to the optical axis. This is in general achieved by using a scanning system, which
allows tilting the laser beam from the optical axis (see Section 2.2.1 for a detailed description).
In a typical experimental setup (Denk et al., 1990), a telescope is inserted between the scanning
system and the focusing lens (objective lens). This telescope has several functions. First of all, it allows
re-conjugating the output of the scanning system with the input of the focusing objective lens. To main-
tain the laser beam centered on the microscope objective back aperture during scanning, it is important
to follow the optical configuration shown in Figure 2.3b. Another crucial role of such a telescope is to
magnify the laser beam diameter in order to fill the objective back aperture and take full advantage of
the numerical aperture (NA) offered by the objective lens (see Section 2.2.5 for a detailed description).
Finally, the telescope de-multiplies (by a factor equal to the magnification) the angle spanned by the
laser beam during scanning, enhancing the scanning sensitivity in the objective focal plane. The mag-
nification of the beam diameter and the de-multiplication factor for the tilting angle are related to the
focal lengths of the two telescope lenses (
f
1
and
f
2
), as follows:
w
w
α
α
f
f
o
i
=
i
o
=
2
1
(2.3)
where
w
o
and
w
i
are the output and input beam diameter, respectively; while α
i
and α
o
are the input
and output laser beam tilting angles, respectively. The value of the tube lens focal length (
f
2
) should be
chosen according to the objective lens used. The most important objective lens producers use tube lenses
of different focal lengths: 200 mm for Nikon, 160 mm for Zeiss, 210 mm for Olympus. Although the
best choice is to use a tube lens with a focal length optimal for the objective lens to be used in order to
minimize aberrations, the values can be slightly changed in a custom-made setup without any dramatic
effect on the image quality.
2.2.2 Laser Source
SHG is a second-order optical process so that the transition probability is extremely low with respect
to other optical linear processes. To excite such a process and to detect a nonnegligible amount of SHG
signal, an extremely high spatial and temporal density of photons is required in the focal volume. Such
high photon density is realized spatially by using a high-NA objective lens (as it will be explained in the
Section 2.2.5) and temporally by using femtosecond-pulsed laser sources. In this section, we describe
the laser sources most commonly used for SHG imaging.
Among femtosecond-pulsed lasers, the most commonly used in SHG imaging is the Ti:sapphire
oscillator which is emitting, depending on the brand and on the model, pulses in the range of 80-200 fs
width at a repetition rate of several tens of MHz (in general between 50 and 100 MHz). Moreover, the
Ti:sapphire laser has a typical emission of up to 5 W tunable in the 700-1000 nm spectral range, cor-
responding to the range in which biological molecules have a significant cross-section for SHG. All
these features make Ti:sapphire the ideal laser source for an SHG microscope. The main drawback is
represented by the cost, which is presently high (typically more than 100 kEuro).
Another femtosecond-pulsed source that can be used for SHG imaging is the Cr:forsterite laser,
which is emitting pulses with width and repetition rate comparable to those of the Ti:sapphire, but in a
different wavelength range (typically between 1150 and 1300 nm) and with a reduced power (typically
between hundreds of mW and a 1-2 W). Even though the Cr:forsterite laser emission spectrum is not
the most suited for SHG imaging (because of the reduced SHG cross-section of biological molecules in
this wavelength range), this laser has the advantage of providing a source not only for second- but also
for third-harmonic generation microscopy (Barad et al., 1997, Oron et al., 2004, Débarre et al., 2006).
Moreover, the cost of such a source is approximately one-half the cost of a Ti:sapphire source.
