Biomedical Engineering Reference
In-Depth Information
the nucleolus, and (3) the nucleolus. Boundaries between ROIs ( Figure 12.12D ) are drawn
manually based on correlations between index tomograms, brightfield images, and widefield
fluorescent images using the nucleic acid stain SYTO (Invitrogen).
Figure 12.12E shows the time dependence of the average refractive index of the three ROIs.
In the nucleolus (ROI 3), a steady increase is observed in average index, which reaches a
stable value about 1.386 within about 2 s. The remainder of the nucleus (ROI 2) exhibits an
average index with similar time course but in the opposite direction, decreasing from about
1.364 to 1.359. It is found that the average refractive index of the nucleus, apart from
nucleoli, to be smaller than that of the cytoplasm. The time dependence of the average
index in nucleus and nucleolus suggests a condensation of nuclear proteins into the
nucleoli. The average refractive index of parts of the cell outside the nucleus (ROI 1) is
largely unaffected by the addition of acetic acid.
Optical scattering properties of a cell are largely determined by spatial variations in refractive
index. To characterize these variations, the standard deviation σ n of refractive index in the
three ROIs ( Figure 12.12F ) is calculated as a function of time. All three ROIs display a
marked increase in refractive index heterogeneity. Remarkably, the three ROIs converge to
similar large values for postacetic acid
σ n , despite a difference in preacetic acid values of
about 40%. The more than twofold greater increase in
σ n for the nucleus and nucleolus
1.39
(E)
(A)
(B)
3
1.4
1.38
n
1.38
1
1.37
1.36
2
1.36
1.34
(C)
(D)
0.010
1
0.008
1
σ n
0.006
2
3
2
0.004
3
0.0
0.5
1.0
1.5
Time (s)
2.0
2.5
Figure 12.12
Refractive index tomograms (cross-section in xy plane) of a HeLa cell during exposure to acetic
acid solution at (A) t 5
m. Image sequence
recorded at 30 frames per second. (D) ROIs described in text. (E) Average refractive index of each
ROI. (F) Standard deviation
0.0 s, (B) t 5
1.3 s, and (C) t 5
2.6 s. Scale bar, 5
μ
σ n of refractive index for each ROI. Solid line: ROI 1. Dashed line:
ROI 2. Dotted line: ROI 3 [25] .
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