Biomedical Engineering Reference
In-Depth Information
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FIgurE 9.10 Hologram reconstruction showing several separate human muscle fibrils. The 3D repre-
sentation shows the assembly of reconstruction planes into the three-dimensional depiction of the sample.
(Reprinted from Masihzadeh, O., Schlup, P., and Bartels, R.A., 2010. Label-free second harmonic generation
holographic microscopy of biological specimens, Optics Express , 18(10), 9840-9851. With permission of Optical
Society of America.)
been reported (Masihzadeh et al . , 2010; Shaffer et al . , 2010b), and holographic SHG imaging of fast-
moving specimens is expected in the near future.
The first of these label-free holographic SHG images of biological tissues was reported by Masihzadeh
et al . (2010), and contained holographic SHG (amplitude contrast) images of several separate human
muscle fibrils. In one experiment (Figure 9.10), they reconstruct the hologram at various depths to obtain
a 3D stack of images, corresponding to cross-sections of the specimens. In another experiment, not pre-
sented here, they also investigate incident-polarization dependence of SHG, and observed that the inten-
sity changes versus the polarization angle, in agreement with previous investigation, performed with
scanning confocal SHG microscopy (Plotnikov et al . , 2006; Tiaho et al . , 2007; Nucciotti et al . , 2010).
That same year, Shaffer et al . studied the SHG of connective tissue in mouse tail dermis, by investi-
gating both amplitude and phase-contrast holographic SHG images (Figure 9.11). While the amplitude
contrast holographic SHG images do not differ much from intensity images obtained by scanning SHG
microscopes, the SHG phase-contrast images are a completely different matter. According to Section
9.5.3, the SHG phase can, in principle, be related to quantitative physical properties, like refractive index
and axial coordinates of SHG. But even if without going into quantitative analyses that may require
some a priori knowledge, the SHG phase is still qualitatively very interesting for investigation of bio-
logical structures. In the following example, we see how SHG phase imaging points to fulfilled phase-
matching conditions in collagen.
9.6.1.1 SHG Phase Matching conditions in collagenous tissues
For some time, it was believed that phase-matching played no role in SHG within collagenous tissues.
Supporting this claim, Kim et al . (1999) predicted, based on values of refractive indices of mammalian
tissues and birefringence properties of collagen available at the time, that phase-matching could not
occur in the visible and near-infrared spectral ranges. That belief held for some time, but was later chal-
lenged (Theodossiou et al . , 2006; LaComb et al . , 2008; Xu et al . , 2010a).
Among the challengers, Theodossiou et al . (2006) pointed out that the refractive indices used by Kim
et al . are group refractive indices for whole tissues (taken from Bolin et al . , 1989), not strictly for col-
lagen, and that therefore, these values do not represent the extraordinary−ordinary interaction which
could be involved in phase-matching considerations. A couple of years later, La Comb et al . explicitly
postulated that the SHG measured in a tissue imaging experiment consists of both (quasi)-coherent
and incoherent scattered components that need to be considered separately for full interpretation of the
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