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of individual slices. Only when examined in series can the extent of the interactions
between mitochondria and LDs be truly revealed.
Figure 8.4
also illustrates one of
the challenges of EM analysis of LDs in a complex tissue, that is, the identification of
a processing method that preserves all organelles and the cellular content in condi-
tions most representative of their natural state and most suitable for imaging. In order
to stabilize the specimen and increase signal strength, sample preparation for
FIB-SEM imaging often requires additional heavy metal staining, a process known
to cause the unwanted extraction of LD stains. Although mitochondria and myofi-
brils are well preserved and stained, staining of LDs in this particular specimen is
not homogenous and as the stain may have been extracted during sample processing.
3D volume reconstruction of the image stack reveals that the stain loss occurs in the
inner core of the LDs (
Fig. 8.5
) and allows to slice through the block of tissue at var-
ious angles so as to view the interaction of LDs and mitochondria in the
xy
,
yz
, and
xz
planes (
Fig. 8.5
). A region of interest can be identified (subvolume) and selected for
segmentation of LDs and mitochondria to examine the spatial arrangement and
interactions between these two organelles.
Figure 8.6
shows the 3D segmentation
of LDs in a subvolume of 31.2
m
3
as indicated in
Fig. 8.5
A (framed in blue line).
m
FIGURE 8.5
FIB-SEM image processing and reconstruction. (A) 3D visualization of a stack of 219 image
slices from a transgenic mouse heart muscle embedded in Durcupan resin. Each slice
thickness is 20 nm. A subvolume (framed in blue line) was selected for segmentation.
(B) Example of one de-noised and shade corrected image slice before segmentation.
(C) Segmentation of lipid droplets (blue) and mitochondria (red) of the image slice shown
in (B).
