Biomedical Engineering Reference
In-Depth Information
As a consequence, a lipid/water volume heterogeneity a few hundred nanometers in size is an efficient
structure for THG by a focused beam. Consistently, lipid droplets have been identified as strong sources
of contrast in THG images of cells and tissues [27]. For the same reason, THG has been reported as an
effective means for imaging myelin in the central nervous system [26,29].
Other dense nonaqueous structures such as mineral deposits, or cell nuclei can usually be detected in
THG images. In contrast, most aqueous solutions exhibit similar values of χ
(3)
[30], implying that THG
from cellular organelles is usually not concentration-sensitive in physiological conditions. We also point
out that, since solvents are optically different from water, the relative visibility of cellular structures is
affected by mounting, index matching, or optical-clearing agents [30,25].
Besides this basic contrast mechanism, the third-order nonlinear susceptibility of a medium is gener-
ally wavelength dependent. In particular, χ
(3)
(−3ω;ω,ω,ω) is altered by 1-photon, 2-photon, or 3-photon
absorption. THG can therefore be resonantly enhanced in absorbing structures. For example, Clay et al
.
[33] identified resonant contributions in the THG signal from hemoglobin, which contribute to the
visibility of red blood cells in THG images. Chloroplasts in plant cells also exhibit strong THG [34],
probably related to chlorophyll absorption properties. Along the same line, Bélisle et al. [35] took advan-
tage of the resonance of hemozoin pigments at the harmonic wavelength to obtain sensitive detection
of malaria-infected cells. Similarly, hematoxylin (an absorbing stain commonly used in histology) has
been shown to enhance the contrast from cell nuclei [36].
3.2.13 one Application of tHG
−
SHG Microscopy: imaging embryo
Development
The previous sections discussed how THG microscopy highlights optical heterogeneities such as intra-
cellular lipidic organelles. At the supra-cellular scale, THG microscopy provides a convenient way to
image the structure of unstained tissues with 3D resolution, while being compatible with SHG or 2PEF
imaging. One field of application that has been explored in recent years is the imaging of embryonic
development in small animal models.
Embryo development involves the spatio-temporal coordination of large ensembles of morphogenetic
processes including collective cell movements and cell divisions. Global imaging of morphogenesis in
complex organisms with subcellular resolution is technically challenging because the shape and opac-
ity of embryos hamper deep imaging. Nonlinear microscopy is attractive for embryo studies because
it provides deep 3D imaging with reduced phototoxicity. Although 2PEF microscopy is the most com-
monly used technique for biology studies, it usually relies on fluorescent protein expression, which can
be challenging to obtain in mutant embryos or at very early stages.
3.2.13.1 Multimodal imaging of Zebrafish embryonic Development
Chu et al
.
[37,38] demonstrated the possibility of imaging developing zebrafish embryos from the cleav-
age stage to the larva stage using THG microscopy without damaging the embryo. They showed that
THG can be combined with SHG imaging of the muscle myofilaments during the larva stage, and even
of the mitotic spindles forming during cell division.
This approach was used by Olivier et al. [21] in conjunction with an optimized scanning scheme to
image all the cell divisions occurring during the first 3 h of development of the zebrafish embryo, and to
reconstruct the cell lineage during the corresponding 10 division cycles.
Representative THG images of zebrafish embryo during divisions are presented in Figure 3.12. Strong
signals are observed near the interface of dividing cells at post-cellularization stages. This signal reflects
the presence of a sizable intercellular space, as corroborated by high-NA THG images of dividing cells
showing locally double interfaces (Figure 3.4d). THG therefore highlights dividing and motile cells,
and provides a direct visualization of cell morphology in the early zebrafish embryo. This remarkable
contrast is compatible with automated cell contour detection [21]. These images also reveal the traffic of
intracellular lipidic vesicles and the dynamics of the vitelline stores (yolk).
