Biomedical Engineering Reference
In-Depth Information
Fig. 6.1
Phase contrast micrographs of (
a
) a keratocyte in a non-polarized and full-moon-shaped
state, and (
b
) that in a polarized crescent-shaped state. Bars, 10
ΚΌ
m
entire cell periphery at the cellular scale, concomitantly with active protrusion and
retraction at the subcellular scale. This organized motility of the full-moon-shaped
keratocyte is expected to include implicitly the underlying interactions as an ensem-
ble of multiple interactions (Satulovsky et al.
2008
). Thus, the full-moon-shaped
keratocyte is a useful cell type to understand the hierarchical, or global and local,
coordination of the actin cytoskeletal system with the top-down image-based
multiscale analysis.
6.2.2
Image Acquisition Condition
A key to success when adopting the top-down approach is the spatiotemporal scales
of image acquisition. The ranges of the scales should be suffi cient to detect the
dynamics of the system being investigated. In this case, the smallest scale that can
be detected is determined by the spatiotemporal resolution of the imaging data, and
the largest by the observation duration and the fi eld of view.
The full-moon-shaped keratocytes exhibit active subcellular protrusion and retraction
with less cellular translocation, which enables acquisition of live cell micrographs
over a long duration with no need of cell tracking device. In addition, these cells have
a broad, fl at lamellipodium consisting of a densely branched actin meshwork
(Svitkina et al.
1997
) with a thickness of 150-200 nm (Laurent et al.
2005
) which
makes it possible to avoid the halo effect under phase contrast microscopy. Phase
contrast microscopy is suitable for high-resolution image acquisition of living
cells by. Compared with fl uorescent microscopy, the advantages of phase contrast
microscopy are less photo-toxic damage and no photo-bleaching effect, which allow
longer observation duration. These advantages in the image acquisition are essential
to reveal the hierarchical properties by the image-based multiscale analysis.
Spatiotemporal resolution with a short timescale (200 ms) and high spatial precision
(80 nm pixel resolution) of a whole single cell, as well as observation duration long
enough to detect slower movement for minutes can be obtained for the full-moon-
shaped keratocyte with the microscopy described in the following section.
