Authors
Ed Luther, Livia Mendes, Daniel Costa, Jiayi Pan, Elena Holden and Vladimir Torchilin
Abstract
Our objective is to develop
multi-functional nanotechnology-based anti-tumor drug delivery systems
for improving efficacy of treatments and reducing undesirable side
effects. The essential part of this process is the development of
un-biased quantitative analytical techniques. We are reporting a
successful validation of a very high content, medium throughput system
in multi-well plates.
We employed a newly developed holographic imaging cytometry system
HoloMonitor® M4 for label-free time-lapse cellular analysis (Phase
Holographic Imaging, Sweden), typically at 5 minute intervals for 48–72
hours.
A low power red laser is split into sample and reference beams to
obtain holograms of cells.
The holograms are unwrapped by proprietary
software into quantitative dark field images, with very precise
calculation of the cell optical thickness.
These images are segmented,
and a vast variety of features are extracted for cellular events.
In our
evaluations we found that cell optical thickness, volume and area have
high correlation with features used in traditional fluorescent
DNA-stained analysis.
We obtain full cell cycle profiles, including the
separation of mitotic cells, cells undergoing mitotic dysfunction, and
apoptotic cells—all in label-free environment.
We recently developed a
novel 4D image display of evaluated fields of view: X position, Y
position, cell thickness coded as brightness over time in the Z
direction.
In these images, the history of the viewing area over time is
displayed, and salient features such as cell proliferation, cell
thickness, and cell motility, contact inhibition, and cell death are
discernable.
As an example, a combinatorial liposomal formulation
containing paclitaxel and a P-gp inhibitor tariquidar was compared to
free paclitaxel in SKOV3 TR (taxol resistant) cells seeded in Petri
dishes. TR cells treated with free paclitaxel presented increased
mitoses, while TR cells treated with the combinatorial preparation
exhibited a complete abrogation of mitotic division.
Cells were frozen
in mitosis due to the polymerization of tubulin, the mechanism of
paclitaxel toxicity.
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