Biomedical Engineering Reference
In-Depth Information
2.1.1 optical table
The mechanical structure of a custom SHG microscope must accomplish several functions. First of
all, it has to be able to damp vibrations and avoid any external mechanical perturbation that can affect
the measurements. Damping is in general obtained by installing the whole system on an antivibration
optical table, which commonly consists of four independent legs that support the weight of a metallic
horizontal breadboard. The legs have a hollow core in which a fluctuating actuator can move vertically
driven by air under pressure provided by an external hydraulic circuit. Recently, optical table produc-
ers have developed a new damping technology based on piezo-electric actuators which are providing
enhanced stability for applications requiring further damping. With this improvement, they offer three
possible solutions for active vibration isolation: air-pressure damping, piezo-electric damping, and also
hybrid systems. Air-pressure damping is generally sufficient for optical imaging applications, while
piezo-electric damping is more suitable for applications requiring higher stability, such as electron or
near-field microscopy.
The horizontal breadboard is made of a damping inner structure sandwiched between two stainless-
steel plates with threaded holes (typically either ¼-20 or M7 thread) on one side in order to allow the
positioning of opto-mechanical components. The two stainless-steel plates are a common feature for
all the optical tables, while the inner structure varies among different models. There are several choices
for the inner structure, including composite laminate, solid material, and lightweight honeycomb.
The choice of construction depends on the type and size of the application, where sizes are typically
120 × 180 cm
2
, 240 cm, or 300 cm (48 × 72, 48 × 96, or 48 × 120″). The optical surfaces with highest
performance are honeycomb core tables, which offer rigidity with a light weight and the possibility to be
extendable up to very large work surfaces. This is in general the most suitable solution for optical imag-
ing applications, such as an SHG microscopy.
2.1.2 Microscope Stand
The microscope stand (intended as the microscope body) provides support to all the optical elements
required for conventional optical microscopy. The most important elements are: the objective lens, the
tube lens, the focusing system, bright field, and/or fluorescence illumination sources, the eyepiece, CCD
mount, and the translational stage on which the sample is mounted. To give to the user the possibil-
ity of adding additional light sources and/or detectors for fluorescence or other imaging techniques,
microscopes are equipped with additional access ports through which it is possible to couple and/or
decouple light to/from the optical axis. Modern commercial microscopes offer solutions that are ergo-
nomic in terms of comfort and versatile in terms of capability to be connected with add-on equipments
(laser light, scanning head, detectors, filters) for various imaging techniques. Anyway, a commercial
microscope is not always the best solution to be adopted for SHG imaging. In fact, while offering a large
set of advantages in terms of simplicity and versatility, they are generally designed for imaging samples
mounted on a standard microscope slide, hence they could have some limitations in imaging
in vivo
or
massive
ex vivo
samples. The choice of the microscope body should thus be driven not only by the opti-
cal imaging technique to be employed, but also by the type of sample the researcher plans to analyze.
Further, commercial microscope stands are generally designed for wide-field fluorescence imaging, so
that different excitation modalities (such as laser scanning; see Section 2.2) and/or peculiar detection
geometries (such as forward detection; see Section 2.3.2.1) cannot be easily implemented. Here, we pro-
vide two alternative solutions to the researchers who want to build their own custom microscope body
without purchasing a commercial product.
2.1.2.1 custom Microscope Stand
In this section, we show an example of a microscope stand built around a custom-made mechani-
cal structure, which is basically equivalent to a commercial microscope skeleton. According to the
