Biomedical Engineering Reference
In-Depth Information
A set of angle-dependent background phase images is acquired with no sample present and
subtracted from the sample phase images to reduce fixed-pattern noise from dust, optical
aberrations, and imperfect optical alignment. The background-subtracted phase images are
used to reconstruct the 3D refractive index of the sample using a diffraction tomography
algorithm based on the Rytov approximation introduced in the previous section.
For cell imaging, cells are dissociated from culture dishes and allowed to attach to
coverslips in normal culture medium (Mediatech DMEM 1 10% fetal calf serum) for about
6 h at 37 C before imaging at room temperature. Coverslips are placed inside a flow
chamber (custom made or Bioptechs FCS2). The culture medium is injected into the
chamber using either a manual syringe or a syringe pump (Harvard Apparatus PHD
22/2000). A valve is used to switch the input to the flow chamber to either a culture
medium containing 0.5% acetic acid (for acetic acid experiments) or a hyperosmolar
phosphate-buffered saline solution (for osmolarity experiments). Using the syringe pump,
the hyperosmotic solution is injected into the chamber at a rate of 1.5 ml/min. Tomograms
are continuously acquired while the new medium is added. Data acquisition is performed
using custom software written in MATLAB (MathWorks, Natick, MA). The 3D diffraction
tomography reconstruction algorithm is performed by custom software written in C.
The rest of the data analysis is performed by custom software written in MATLAB. Using
a computer running Windows XP 64-bit edition with an Intel Core 2 6600 processor
running at 2.4 GHz and 2.93 GB of RAM, the computation time required to construct a
single tomogram from 150 interferogram images is approximately 5 min.
12.4.2 Results
To demonstrate the instrument's capabilities, changes in structure of a single cell are
monitored during exposure to acetic acid. Acetic acid is widely used during colposcopy to
identify suspicious sites on the cervix, due to its whitening effect in precancerous lesions
[31] . Previously, it was shown that acetic acid causes an increase in refractive index
inhomogeneity throughout a cell and increases the index of the nucleolus [13] . However,
the time course of these changes remains unclear due to a limited temporal resolution.
To examine these changes in refractive index in detail, tomograms of a HeLa cell are
recorded while the cell is exposed to a new medium containing acetic acid. Almost all
changes in the cell structure were found to occur within a 2.75 s interval. Figure 12.12A
C
shows xy slices through the center of the cell at the start, midpoint, and end of this
interval. An increase in refractive index heterogeneity is observed throughout the cell and
the index of the nucleolus increases dramatically. To assay the effects of acetic acid on
different components in the cell, we partitioned the xy slices into three distinct regions of
interest (ROIs) approximately corresponding to (1) the region between the cell boundary
and nuclear boundary, (2) the region enclosed by the nuclear boundary but not including
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