Biomedical Engineering Reference
In-Depth Information
(a)
Filling
(b)
Underfilling
1/
e
2
1/
e
2
Beam
waist
FIgurE 2.9
Beam size adjustment relative to the objective's back aperture. (a) Filling of the back aperture with
an expanded beam. (b) Underfilling of the aperture, the transmitted power is maximum but the spatial resolution
is reduced. The beam waist size in the focal volume is reproduced in the inset.
2.2.6 Polarization Scanning
An important feature in an SHG microscope is represented by the capability to vary the polarization
of the exciting laser beam (Stoller et al., 2002a,b, Tiaho et al., 2007, Matteini et al., 2009, Nucciotti
et al., 2010). In fact, the SHG intensity in far-field strongly depends on the mutual orientation between
the molecular intrinsic hyperpolarizability and the direction of oscillation of the exciting field (Mertz
and Moreaux, 2001). By using this property, it is possible to measure the angular distribution of hyper
Rayleigh scattering (HRS) emitters in the focal volume, which is in turn related to molecular structural
organization of the specimen under investigation. The measurement is usually performed by acquiring
multiple images with different polarization orientations. The result is an image in which a polarization
profile is embedded in each pixel. Such a profile contains the information on the angular distribution
of HRS emitters within the focal volume. A fitting procedure by using a proper theoretically calculated
function allows to extract the parameters describing the structural organization within each pixel (Sun,
2005, Plotnikov et al., 2006, Tiaho et al., 2007, Nucciotti et al., 2010).
The polarization scanning equipment is usually placed in a section of the microscope in which the
beam is collimated, as close as possible to the objective lens. As a general rule, the equipment for polar-
ization scanning comprises three main parts (Figure 2.10a).
• A first
polarizer
inserted in the beam path in order to make the polarization linear. In fact, even
if the laser output is polarized, the beam travels through several optics which have detrimental
effects on the polarization. A good linear polarization has to be obtained in order to provide the
required input condition for the following component: a quarter wave plate.
• A
quarter wave plate
is inserted in the optical path and properly oriented for generating a circular
polarization.
• The last element is a
rotating polarizer
for scanning the sample with a linear rotating polarization.
The polarizer can be rotated by means of a rotating opto-mechanical component equipped with
a stepping or a DC motor.
It has to be noted that, if higher polarization scanning speed is required, the last element (the rotating
polarizer) can be substituted with an active element such as an electro-optical modulator (EOM) which
is providing a much faster response with respect to a rotating polarizer, which offers a scanning speed
limited by its inertia. It is also important to note that the light beam coming out from the quarter wave
plate must have a perfect circular polarization in order to acquire accurate polarization SHG profiles.
