Biomedical Engineering Reference
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FIgurE 15.2 ( See color insert .) SHG/2PEF imaging of lung fibrosis in bleomycin-treated mice. SHG (green: col-
lagen) and 2PEF (red: elastin, macrophages) signals excited with 50 mW excitation power at 860 nm. Yellow color:
SHG/2PEF colocalization. (a) Fresh unlabeled fibrotic lung (day 14), at 42 μm under the pleura, evidencing the het-
erogeneous fibrosis distribution; (b) 3D reconstruction of the SHG signal from the underlined area in (a), within the
tissue (b1) and in the pleurae (b2); (c) unstained histological sections embedded in paraffin, from (c1) control, (c2)
day 3, (c3) day 7, and (c4) day 14 bleomycin lungs. Bleomycin treatment induces (i) inflammation, which decreases
after day 7, as evidenced by the 2PEF signal, and (ii) fibrosis, evidenced by the SHG signal detected as early as day
3. Scale bar: 100 μm. (From Pena, A.-M. et al. Three-dimensional investigation and scoring of extracellular matrix
remodeling during lung fibrosis using multiphoton microscopy. Microsc. Res. Tech. 2007. 70:162-170. Copyright
Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.)
15.3.2.1 3D capability
Like other multiphoton microscopies, SHG microscopy offers 3D capability that enables the visualiza-
tion of the 3D architecture of collagen fibrosis. Other techniques are either restricted to 2D sections
(histological or immunochemical labeling) or show limited specificity to fibrillar collagen (reflectance
confocal microscopy or optical coherence tomography). SHG microscopy is therefore the only tech-
nique that enables highly specific 3D imaging of intact collagen tissues.
15.3.2.2 Highly contrasted images/Sensitivity
The high specificity of SHG microscopy to fibrillar collagen results in an excellent signal-to-noise ratio
in the SHG images and enables sensitive measurements of the fibrosis distribution. Furthermore, SHG
scales quadratically with the density of aligned collagen molecules within the focal volume, as explained
in the former section (see Figure 15.1). It results in highly contrasted images compared to conventional
techniques that all scale linearly with the density of (aligned) collagen molecules. This feature was
recently illustrated in collagen liquid crystalline samples where both the SHG and the 2PEF signals from
collagen were recorded (Deniset-Besseau et al. 2010). The SHG signal intensity unambiguously displayed
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