Biomedical Engineering Reference
In-Depth Information
generally obtained by using the so-called SHRIMP (second-harmonic radiation imaging probes). These
SHRIMPs are generally nanocrystals that can be functionalized for selective labeling of biological sam-
ples. The SHG properties of several kinds of nanocrystals have already been reported: ZnO (Prasanth
et al., 2006), KNbO
3
(Nakayama et al., 2007), BaTiO
3
(Hsieh et al., 2009). For example, a measurement of
the SHG cross-section for BaTiO
3
nanocrystals gave a value in the order of 10
4
GM (Hsieh et al., 2009). It
has to be considered that typical cross-sections for two-photon fluorescence probes, for example, EGFP,
are in the order of tens of GM (Heikal et al., 2001).
2.3 the Detection System
In this section, we analyze different optical detection modalities and geometries. In detail, we describe
two detection geometries: forward detection and backward detection. The former is the most used, since
SHG is mainly emitted in the forward direction. The latter is commonly used when imaging a sample
with thickness large enough to prevent forward detection. In the last part, several optical filtering and
detector solutions will be reviewed.
2.3.1 optical Detection Modalities
There are two different detection modalities in laser scanning microscopy: de-scanned and non-
de-scanned. The de-scanned mode is analogous to the confocal geometry: SHG light passes through the
scanning head before being decoupled from the laser path and detected. In the non-de-scan mode, SHG
light is decoupled from the laser path immediately behind the objective. The main difference consists
of the fact that in de-scan mode the focused SHG light into the detector maintains the same position
during scanning while in non-de-scan mode the focused SHG light scans across the detector. For deep
tissue imaging, the non-de-scan solution (allowing the closest proximity of the detector to the objective
lens) is essential to minimize the loss of multiple-scattered SHG photons from the tissue.
2.3.2 Detection Geometry
2.3.2.1 Forward Detection
Forward detection is the most common detection geometry used in SHG microscopes. Such detection
geometry is preferably used with thin samples, such as cells (Campagnola et al., 1999, 2001), microtu-
bules (Dombeck et al., 2003), cellular membranes (Bouevitch et al., 1993, Peleg et al., 1999, Moreaux
et al., 2000a,b, Sacconi et al., 2005), and muscle fibers (Both et al., 2004, Boulesteix et al., 2004, Plotnikov
et al., 2006, Nucciotti et al., 2010). In fact, as known from SHG theory (Moreaux et al., 2001, Mertz et al.,
2001), SHG is a coherent process preserving both energy and momentum. Therefore, SHG is mainly
directed in the same direction as the exciting wave vector. As shown in Figure 2.14a, the ratio between
forward and backward emitted SHG is ranging from 1 to 10
12
, depending on the angle between the
single HRS dipolar axis and the optical propagation axis (Zipfel et al., 2003a). More in detail, as shown
in Figure 2.14b, the forward emitted SHG light has a characteristic spatial pattern with two lobes form-
ing an angle with the optical axis θ
peak
. This angle is related to the phase anomaly and hence to the NA
of the excitation objective (Moreaux et al., 2000b).
In agreement with this description, several considerations must be done regarding the collection
objective lens. In principle, the ideal optical geometry is realized using a collection objective with the
same NA of the excitation objective. If we are using a collection objective with lower NA, a substantial
loss of SHG photons will occur. On the other hand, in a real experimental setup, it is preferable that the
NA of the collection objective be slightly higher than the excitation one, so as to prevent photons leakage
due to a inevitable misalignment of the objectives confocality during experiment (e.g., in axial scanning).
This optical configuration is maximizing the detected signal. As a general rule, objective lenses guar-
antee higher collection as the NA increases, so that the optimal solution should be, in principle, to
