Biomedical Engineering Reference
In-Depth Information
represent an obstacle for the researcher. An alternative solution, with less complexity, much less time-
consuming, upright geometry, and with the potential to be customizable is the use of a vertical optical
breadboard, as described in the following section.
2.1.2.2 optical Breadboard Solution
To have a customizable microscope without the complexity related with the solution presented above,
an optical breadboard can be used as a microscope stand. For example, the solution presented by Cicchi
et al. (2008) consists of a two-photon fluorescence/SHG custom-made microscope in which all the optics
are fixed onto a stainless-steel honeycomb breadboard vertically mounted on an antivibrating optical
table. A picture of this experimental setup is shown in Figure 2.2. The breadboard can host all the micro-
scope components, including scanning head, scanning lens, tube lens, objective, sample stage, optical
filters, and detectors.
The breadboard allows mounting all the optical components required for the experiment with easy
interchangeability of the optical configuration. In comparison with the previously described solu-
tion, realizing such instrument is much simpler because the number of mechanical parts is drastically
reduced, while the customizability remains unchanged. The main drawback of this solution is that it is
harder to optically align all the system on a vertical plane.
An alternative solution is represented by mounting the microscope onto a horizontal optical bread-
board, which offers an easy alignment procedure but it has the drawback of making an
in vivo
measure-
ment on cells or tissues extremely hard because the sample has to be placed vertically. Both problems
(ease of alignment and sample positioning) can be circumvented by aligning the system with the bread-
board placed horizontally and then, once aligned, mount it in vertical onto the optical table. A couple
of mirrors placed very close to the breadboard is strongly recommended in this configuration in order
to have all the degrees of freedom on beam tilting required for an optimal optical coupling of the laser
beam with the vertical optical system.
3
7
8a
2
6
8b
4
5
1
FIgurE 2.2
The figure shows the laser beam [in dark gray, coming from the bottom right side of the panel (1)]
that is first scanned by two galvanometric mirrors (2), then expanded by a telescope (3), and finally focused by the
objective (4) onto the specimen (5). The emitted light (light gray) is separated from the exciting beam by a first
dichroic mirror (6) and then split by a second dichroic mirror (7) in two distinct chromatic components (TPEF and
SHG). Two PMTs detect the split emissions (8a,b). (From Cicchi, R. et al. 2008.
Appl. Phys. B
, 92: 359-365; Allegra
Mascaro, A. L., Sacconi, L. and Pavone, F. S. 2010.
Front. Neuroenerg
., 2: 21.)
