Biomedical Engineering Reference
In-Depth Information
(b)
(a)
THG
y
Exc.
2PEF
x
Cells
THG
SHG
Excitation
z
2PEF
400
800
1200 (nm)
THG
SHG
THG
SHG
2 PEF
(c)
(d)
(e)
Yolk
FIgurE 3.12
Multimodal nonlinear imaging of a zebrafish embryo. (a) THG image of a zebrafish embryo during
initial cell divisions. Scale bar 200 μm. (b) Geometry and wavelengths involved for multimodal THG−SHG−2PEF
imaging. (c-e) Simultaneous THG−SHG−2PEF image of dividing cells in a H2B-mCherry embryo. Scale bar
20 μm. (From Olivier N et al. 2010. Cell lineage reconstruction of early zebrafish embryos using label-free nonlin-
ear microscopy.
Science
329:967-971. Reprinted with permission of AAAS.)
Combined THG/SHG imaging can be used to detect a number of key events during cell divisions.
SHG reveals the formation of mitotic spindles, providing a specific marker of cell divisions. The SHG
intensity exhibits a nearly Gaussian temporal behavior with a maximum intensity corresponding to the
anaphase, permitting the measurement of division timings and cell cycle duration with high precision.
Moreover, THG, SHG, and 2PEF signals from red fluorescent proteins can be produced and detected
simultaneously. Figures 3.12c through 3.12e show an example of multimodal THG−SHG−2PEF imag-
ing of H2B-mCherry mutant embryos, where the 2PEF signal is used to follow the chromatin distribu-
tion along with THG−SHG signals [21].
3.2.13.2 tHG
−
SHG imaging of other embryos
Similarly, THG−SHG microscopy has also been shown to be an effective approach for providing morpho-
logical images of developing embryos in other species: drosophila [39], mouse [40,41],
Caenorhabditis
elegans
[42], and so on. We note that, unlike in zebrafish embryos, in these other species the THG
images usually reveal the intracellular organelles rather than the cell contours.
3.3 tHG Microscopy of Anisotropic Media
In the previous section, we discussed THG by simple isotropic media. In this section, we will consider
more complex media. We will first discuss the sensitivity of THG to tensor symmetries and to the
incident polarization, and discuss THG by anisotropic and birefringent media. We will then out-
line the possibility of quasi-phase matching from axially periodic samples. Finally, we will illustrate
the application of THG−SHG imaging to a highly organized biological medium, namely the human
cornea.
3.3.1 Field and tensor Symmetries
The nonlinear susceptibility χ
(3)
relating the induced third-harmonic polarization to the incident fields
is generally a fourth-rank tensor. However, before discussing the χ
(3)
tensor properties, we point out
that the excitation field distribution is also intrinsically vectorial, even in the case of an incoming linear
polarization. For example, the
E
z
component of the electric field of a tightly focused Gaussian beam may
not be negligible, as illustrated in Figure 3.13.
