Biology Reference
In-Depth Information
remove a thin layer of specimen and then record the image of the newly exposed
surface. This milling and imaging sequence can be repeated using preset parameters
in an automated fashion, thus allowing the collection of stacks of images for a whole
cell or large volumes of tissue. The potential of this technique is illustrated by recent
publication of 3D image analyses of a whole yeast cell (
Wei et al., 2012
), mammalian
cell (
Bennett et al., 2009; Heymann et al., 2006, 2009
), and brain tissue (
Knott,
Marchman, Wall, & Lich, 2008, Knott, Rosset, & Cantoni, 2011
). Furthermore, it
has been used to demonstrate differences in the volume and surface curvature of
mitochondria in diseased versus normal liver tissue, suggesting it may be possible
to apply this technique in a clinical screening situation (
Murphy et al., 2010
). We
present here our preliminary data on the 3D analysis of LDs in resin-embedded
mouse heart tissue. Since this work is still in progress, the purpose of this section
is not to provide the reader with a detailed protocol but to demonstrate the potential
of this newmethodology and to stimulate discussion with respect to the pros and cons
and the challenges of this 3D imaging method.
To pilot the FIB-SEM application to the study of cardiac LDs, we used hearts
specimen isolated from mice with cardiac myocyte targeted Plin5 expression
(MHC-Plin5) (
Wang et al., 2013
). Increased perilipin 5 expression in these trans-
genic mice leads to storage of excessive triacylglycerol in LDs and cardiac steatosis
(
Pollak et al., 2013; Wang et al., 2013
). A total of 219 FIB-SEM image series of heart
muscle from a MHC-Plin5 transgenic mouse were collected at 20 nm thickness per
slice using a Zeiss Auriga cross beam FIB/SEM platform. In
Fig. 8.4
, we present
selected images progressing through an
600 nm depth of the heart muscle speci-
men. These series of images clearly demonstrates the limitations of 2D viewing
FIGURE 8.4
Selected FIB-SEM image slices. From slice 54 to slice 83 progressing through 600 nm
depth of the heart muscle specimen are presented. The fusion event between two LDs (white
arrow) will most likely be missed by conventional TEM.
