SHG Imaging of Collagen and Application to Fibrosis Quantization - Second Harmonic Generation Imaging - page 355

(a) (b)

D0 D0

(P) (M) (C)

D28 D28

(C)

(P) (M)

D49 D49

(P) (M)

(C)

(e)

4

(c) (d)

D49

2PEF
SHG

Exc.

D28

Y

Control

3

C

1/4

P M

X

1/4

2

Z

Z

1/2

1

0

200 µm 0.00 0.20 0.40 0.60

SHG density in cortex (%)

0.80 1.00

FIgurE 15.3 ( See color insert. ) Multiphoton images of fibrotic murine kidney tissue. (a) SHG/2PEF and (b)
segmented SHG images of coronal renal section from a control, 28-days, and 49-days Angiotensin II infused mouse
showing renal papilla (P), medulla (M), outer cortex (C), and arcuate arteries along the boundary between cortex
and medulla. SHG (green color) reveals collagen fibers in the tubular interstitium and in the arterial adventitia, and
2PEF (red color) underlines tubules. These 4.8 × 2.4 mm 2 images are obtained by stitching 10 × 20 laser scanned
images (dimension: 270 × 270 μm 2 , pixel size: 0.8 × 0.8 μm²) acquired sequentially by moving the kidney sample
with a motorized microscope stage. Scale bar: 500 μm. The white lines underline segmentation used for SHG scor-
ing (excluding the artery region and the borders). (c) Scheme of the laser scanning multiphoton microscope show-
ing epidetection of z -stacks of SHG/2PEF images and kidney coronal slicing for multiphoton microscopy. (d) 3D
reconstruction showing interstitial fibrosis (270 × 270 × 40 μm 3 with 0.4 × 0.4 × 0.5 μm 3 voxel size). (e) Automated
SHG scoring of cortical fibrosis plotted as a function of a semi-quantitative estimate of interstitial damage for con-
trol and hypertensive mice. Both scores are correlated ( τ = 0.68, p < 0.01). (From Strupler, M. et al. 2008. J. Biomed.
Optics 13:054041. With permission of SPIE.)