Tomographic Phase Microscopy (TPM) - Biomedical Optical Phase Microscopy and Nanoscopy - page 231

of the nucleoli, n 1.38, is larger than that of the rest of the nucleus. This is contrary to the

widely cited claims that the refractive index of the nucleus as a whole is higher than that of
the rest of the cell [28] . Similar results were obtained for cultured HEK 293 cells, B35
neuroblastoma cells, and primary rat hippocampal neurons. All cells imaged contained
many small cytoplasmic particles with high refractive index, which may be lipid droplets,
lysosomes, vacuoles, or other organelles.

To demonstrate tomographic imaging of a multicellular organism, the nematode
Caenorhabditis elegans , which is paralyzed with 10 mM sodium azide in nematode growth
medium (NGM) buffer, is imaged in the same solution. Overlapping tomograms are created
and the resulting data assembled into a mosaic ( Figure 12.4B ) and compared with its
brightfield counterpart ( Figure 12.4A ). Several internal structures are visible, including a
prominent pharynx and digestive tract.

12.3.3 Data Processing by the ODT

Using the set of phase and amplitude images taken at various angles of illumination, the
diffraction tomography algorithm described in Section 12.2.2 can be applied for the 3D
reconstruction. Given a quantitative phase image φ ( x , y ; θ ) and an amplitude image A ( x , y ; θ )
taken at each illumination angle, θ , the complex E-field at the image plane is constructed by
U ( x , y ; )e i φ ( x , y ; θ ) . The measured field image is composed of the phase change

induced by the sample and the phase ramp introduced by the tilted illumination.
A corresponding set of images U bg ðx; y; θÞ 5 A bg ðx; y; θÞ

θ ) 5 A ( x , y ; θ

e i k x 0 x 1 i k y 0 y taken when no sample is
present provides the background field, which can be considered as the incident fields.

(A)

(B)

Figure 12.4
(A) Mosaic of brightfield images of a juvenile C. elegans nematode. (B) Mosaic of xy slices of
index tomograms through center of worm. The color bar indicates the refractive index at
λ 5 633 nm. Scale bar, 50 μ m [13] .