Biomedical Engineering Reference
In-Depth Information
• The laser source (Ti:sapphire) providing femtosecond pulses. See Section 2.2.2 for a detailed
description of suitable laser sources for SHG microscopy.
• The power adjustment system (Section 2.2.3), made by a rotating half-wave plate (λ/2), (remotely
controlled from PC), and a polarizing beam-splitter cube (PBS).
• The scanning system (Section 2.2.4), made with two orthogonal galvo-mirrors (
G
x
and
G
y
) opti-
cally relayed by a telescope (L1,L2) with magnification equal to 1. A detailed description of the
basic principle of a scanning system is provided in Section 2.2.1.
• The telescope (L3,TL) for magnifying the beam size and de-multiply the scanned angle. See
Section 2.2.1 for a detailed description of the function accomplished with this optical configura-
tion. Generally, the magnification obtained ranges from 3 to 5.
• A polarization scanning system made with: a polarizer in order to make the polarization linear;
a quarter wave plate for generating a circular polarization from a linear one; a rotating polarizer
for illuminating the sample with a linear rotating polarization. See Section 2.2.6 for a detailed
description of the polarization scanning system.
• The excitation objective lens (Exc). See Section 2.2.5 for a more detailed description.
• The collection objective lens (Coll). See Section 2.3.2.1 for a more detailed description of the for-
ward detection geometry.
• The optical filtering system, composed of a dichroic mirror (DM), a laser-blocking filter (BF), and a
narrow band-pass filter (BP). See Section 2.3.3 for a detailed description of the optical filtering system.
• The detection system (Section 2.3), constituted by a collecting lens (L4) and a PMT. It has to be
noted that the focus of the SHG light detected by the PMT is scanned across the detector during
scanning so that a detector with large sensitive area is necessary in this configuration, as previ-
ously described in Section 2.3.4.2.
2.4.2 Backward Detection
This optical configuration is generally used in SHG imaging of massive samples, such as bulk
ex vivo
excised tissues (biopsies) or
in vivo
experiments. The backward detection geometry can be performed in
both non-de-scan and de-scan mode.
2.4.2.1 non-De-Scan Mode
The main differences with respect to the previous detection scheme are:
• The absence of a collection objective. The excitation objective serves for both excitation and
collection.
• The position of the detection system (L4, PMT) and the optical filtering system (DM, BF, BP). In
this optical configuration, these two systems are placed immediately behind the excitation objec-
tive, as close as possible to the objective back aperture.
As in forward detection, in non-de-scan mode, the focus of the SHG light detected by the PMT is
scanned across the detector during scanning, so that a detector with large sensitive area is necessary in
this configuration, as previously described in Section 2.3.4.2.
It has to be noted that the backward scheme strongly limits the use of a polarization scanning system.
In fact, if the polarization scanning system is placed before the dichroic mirror (DM), the birefringent
coating of DM can affect the intensity uniformity during polarization scanning (see Section 2.2.6). On
the other hand, if the polarization scanning system is placed between the dichroic mirror (DM) and the
objective lens, the polarization optics will cause a drastic reduction of the detected SHG signal.
2.4.2.2 De-Scan Mode
The main difference with respect to non-de-scan mode consists in placing the optical filtering system
(DM, BF, BP) and the detection system (L4, PMT) before the scanning system (
G
x
, L1, L2,
G
y
).
