Tomographic Phase Microscopy (TPM) - Biomedical Optical Phase Microscopy and Nanoscopy - page 247

distribution of nucleolus when the incident beam is parallel to the optical axis. Oscillations in
angular distribution are much coarser than those of the whole cell, and the asymmetry of the
light scattering distribution was as expected from the asymmetry of the shape. This is the first
time in which light scattering of a single nucleolus in an intact living cell has been assessed.

A similar procedure was applied to assess the scattering of the nucleus. In the
nucleolus-free tomogram ( Figure 12.16B ), there are three major compartments causing light
scattering: cell boundary, particles in the cytoplasm, and the nucleus. The cell boundary and
particles in the cytoplasm were eliminated at every section by setting the index of the
surrounding area of the nucleus as the average index of cytoplasm ( Figure 12.16C ). This
modified tomogram contains only the scattering contribution of the nucleus. Using the
Rytov approximation, nuclear scattering was directly calculated from this modified
tomogram ( Figure 12.16F ). The oscillations in angular distribution are finer than those of
the nucleolus, as expected.

Next, we studied the relative strengths of the scattering from the different organelles.
In order to determine the morphology of the specific organelles such as nuclei from the
light scattering measurement of the entire cell, the measurement must be sufficiently
sensitive to account for baseline scattering from the whole cell. The knowledge of the

(A) (B) (C)

5 μ m 5 μ m 5 μ m

(D) (E) (F)

Figure 12.16
Cross-section of 3D refractive index tomograms and 2D angular scattering maps. (A) Original
tomogram section of HT29 cell. (B) Tomogram section with nucleolus replaced with random
nuclear index values. (C) Tomogram of the cell nucleus. The color bar indicates refractive index at
wavelength of 633 nm. Scale bars, 5 m. (D) Angular scattering intensity of the original cell

tomogram. (E) Scattering intensity of the nucleolus. (F) Angular scattering of the nucleus. The
color bar indicates intensity in base-10 logarithm with arbitrary units [39] .

μ