Biomedical Engineering Reference
In-Depth Information
thanks to development of ultrafast lasers: without ultrashort pulse lasers, there would be no nonlinear
microscopy.
Nevertheless, the development of ultrafast laser sources is probably more important to holographic SHG
than it is to other nonlinear microscopy techniques. The many reasons for this can all be summarized in
one: holography is a nonscanning technique. This means that to produce an image, the light source must
illuminate the entire field of view, as it is the case with bright-field microscopy. Therefore, to deliver the
same peak power density to the specimen that of a confocal scanning microscope, the laser source for
holography has to be much more powerful, in terms of mean power. Very high-power lasers would make
possible to illuminate a large region of the specimen, thus providing a large field of view, while keeping the
local peak power densities similar to those used in scanning microscopy, to generate comparable signals.
Provided very fast digital cameras, the maximum frame rate becomes limited by the quantity of
second-harmonic generated photons that produce the hologram contrast. Therefore, the more powerful
the laser source, the larger the possible field of view and the higher the maximum frame rate. At least,
until photo-induced damage becomes an issue.
Photo-induced specimen damage can be a problem in nonlinear microscopy. The weak signals of inter-
est are an incentive to increase the laser power. There is however a limit to the energy one can deliver
onto the specimen without damaging it. That limit varies from specimen to specimen and depends on
the wavelength of the laser. The specimen damage threshold is related to the total absorbed energy per
unit volume per unit of time. As typical duration of ultrafast laser pulses are much smaller than the char-
acteristic time of bio-physical phenomena, for example, heat dissipation, this limit can be reported to
a total absorbed energy per unit volume per pulse, assuming that the time lapse between two pulses is
large enough to allow the specimen to return to its equilibrium state. As the absorption depth is generally
limited by the specimen thickness or extinction coefficient, these parameters can be reported in terms
of energy per surface. For a given pulse duration, the striking conclusion to this is that delivering more
energy over a proportionally larger surface maintains both the SHG signal per unit surface and the risk of
photo-induced damage constant.
For the same pulse duration and energy per pulse, the SHG intensity scales linearly with the repetition
rate. The higher the repetition rate, the stronger the SHG signal. When selecting a laser for holographic
SHG microscopy, the thumb rule for the repetition rate thus is: the higher the better, as long as the time
between two successive pulses is long enough to allow return of the specimen to equilibrium state. As
an indicator, 80 MHz repetition rate lasers generally do not pose a problem. If also specimen-safe, a
2.0 GHz laser with comparable pulse duration and energy per pulse would yield a 25 times enhancement
of the SHG signal.
The amplitude of the second harmonic generated varies with the square of the amplitude of the
instantaneous electric field induced by each laser pulse. For given energy per pulse, the shortest the pulse
duration, the higher the instantaneous electric fields and, in turn, the strongest the generated SHG. Of
course, as the pulse duration becomes very short, its spectrum broadens and its temporal coherence
declines—see Table 9.1. The consequence of this is a much reduced area on the detector where interfer-
ence occurs between the object and the reference wave. This effect, called beam walk-off, could pose
a serious problem for holography. Fortunately, different physical implementations of the holographic
interferometer have already been proposed to overcome the beam walk-off problem (Maznev et al
.
, 1998;
Ansari et al
.
, 2001). With such implementations, it is expected that the large spectrum broadening that
comes with much reduced pulse duration would not pose a problem to the holographic scheme.
9.3.2.2 Detector
Being a nonscanning imaging technique, holographic SHG requires a full 2D digital sensor, typically a
CCD or CMOS camera. The nature of holography and of SHG imposes some requirements on the speci-
fications of such sensors. When looking for a digital sensor, specifications like frame rate, exposure time,
and gain range (and their increments) immediately come to mind. However, apart maybe for fluores-
cence imaging that requires very long exposure times, these specifications are not so much related to the
