Biomedical Engineering Reference
In-Depth Information
the reflection setup we provide here should be viewed as a setup proposal and not as the description of a
state-of-the-art microscope for backscattered second-harmonic signal.
The two implementations of Figure 9.3 are based on Mach−Zehnder interferometers which, because
they are of very simple design and do not have folded optical path like Michelson's, are very convenient
for generation and shaping of the reference and object waves. In fact, they are up to now the only type
of interferometer reported for holographic SHG microscopes. In the setups of Figure 9.3, a pair of beam
splitters are used to separate the laser beam into the reference and object arms, and to recombine them
in the infinity space of the microscope objective.
A variable optical delay line (DL) in the reference arm makes possible to match very precisely the
optical path of both arms, so that
o
and
r
may interfere on the digital sensor (CCD). Coherence lengths
of femtosecond lasers are typically in the order of tens of micrometers so temporal coherence overlap
has to be very precisely adjusted.
The second-harmonic reference wave is generated by focusing the beam in a frequency doubler crystal
(FDC). It is then collimated and expanded by pair of lenses with different focal lengths. A pinhole may
be introduced in the focal plane of the beam expander (BE) to clean the beam of any high-frequency
content. A similar beam expander is located in the object arm to match the beam size to the clear aper-
ture of the condenser lens (CL).
The condenser lens very loosely focuses the beam on the specimen. Since holographic SHG micros-
copy is a bright-field technique, the illumination must cover the entire field of view. This constitutes a
major difference with scanning SHG microscopes, where the illumination is focused on a very tight
spot. There are two very simple ways to ensure that the illumination is large enough. One is to focus the
illumination at some distance below or above the specimen. The other is to use a condenser lens of long
focal length and/or small diameter that will make a diffraction limited spot matching the field of view.
This last method is generally preferable, since it makes the specimen lie in the waist of the beam, where
the wavefront is more or less planar. This becomes even more important when investigating the second-
harmonic response of the specimen with regard to the incident polarization. Although not illustrated
in Figure 9.3, a Köhler illumination could also be used, since, after all, holographic SHG microscopy is
based on a bright-field illumination.
To record holograms, it is preferable to have the reference wave (instead of the object wave) sub-
tending the off-axis angle with the optical axis. This way, the object wave can propagate along the
optical axis and hit the sensor perpendicular to its surface. Such arrangement is not only easier to
align, but also makes the image plane parallel to the sensor surface. In the proposed implementa-
tions, an infinity-corrected objective is used and its tube lens serves both for forming the image of
the object and to collimate the reference wave. Laterally moving the center of curvature (CC) of the
reference wave gives it an off-axis angle upon collimation by the tube lens. One advantage is that the
recombining beam splitter is located in the infinity space of the objective, where the diffraction orders
propagate parallelly.
Finally, for both reflected-light and transmitted-light implementations, the digital sensor (CCD) is
positioned at some distance before the system image plane, to record out-of-focus SHG holograms in
the Fresnel configuration.
9.3.2 Key elements of a Holographic SHG Microscope
This section discusses the key elements needed in a typical holographic SHG microscope. These elements
include the laser source and the detector, essential to any digital holographic setup, but also elements spe-
cific to holographic SHG imaging like, for example, optical delay lines and frequency doubling crystals.
9.3.2.1 Ultrafast Laser Source
The most important element of a holographic SHG microscope is, without any doubt, its laser source.
Putting it this way is probably an understatement, since nonlinear microscopy, in general, exists only
