Imaging Provides New Insights in
By combining backscatter image sequence acquisition and deconvolution, a new
high-isotropic resolution imaging method has been developed for cell biologists.
Cell biologists need high-resolution 3-D imaging to understand the structure- function relationships of organelles and other structures in cells, and the connectivity and organization of cells in tissues. Many
of these structures are too small to be seen clearly in a light microscope and require the higher
resolving power of an electron microscope.
Scanning electron microscopes (SEM) are
becoming increasingly popular for 3-D studies in neurobiology due to new physical slicing
techniques. When using these approaches, the
section thickness often limits the resolution in
the z-direction. For instance, Drosophila brain
tissue exhibits neurites less than 40 nm, which
could cause a misinterpretation of connectivity
if cuts were made at the same order of magnitude.
Serial slicing methods based on diamond-knife cutting are, however, reaching practical
limitations in terms of achievable z-resolution
and voxel isotropy. This is mainly due to the
difficulty of cutting sections thinner than 15
nm with the desired consistency. While focused
ion beam (FIB) serial block face imaging can
improve the z-resolution to 5 nm, this technology is restricted due to the total volume of
material that can be processed.
Figure 1: Mouse brain tissue studied in 3-D with
ThruSight technology. Image: FEI Co.
Scientists at FEI, Hillsboro, Ore., invented
the new ThruSight method that achieves high-isotropic resolution by a combination of backscatter image sequence acquisition and deconvolution (DC) (Figure 1). ThruSight is built
on improved understanding of beam-sample
interaction for classically prepared resin-embed-ded samples. Monte Carlo simulation tools
and experimental observations show that these
materials exhibit high linearity, allowing for the
use of image formation models based on linear
convolution. Furthermore, the point spread
function (PSF) of backscatter electrons (BSE) in
these materials appears to be well confined laterally for the typically used primary energies.
As the range of penetration in the sample is
dependent on the energy of the primary beam,
acquiring an image sequence with increasing
landing energies leads to the acquisition of
images from increasing depth. These images
contain overlapping volume information from
which 3-D layers can be extracted using DC
algorithms (Figure 2).
The good lateral confinement of PSFs allows
for restricting the DC to the z-axis making it
similar to a source separation task. As the structure of the PSF is difficult to obtain experimentally, it is a variable in the resulting blind DC
problem. Efficient iterative methods allow for
the recovery of both the depth layers and PSFs.
To verify the reconstruction results FEI com-
bined this technique with classical FIB-SEM
serial block imaging using the through-the-lens
detector in BSE mode on a Helios NanoLab 650
DualBeam. The z-resolution was controlled by
switching the primary beam energy in small
steps. The comparison with a high-resolution
FIB reconstruction showed identical structures
on the studied samples proving the reliability
of the 3-D technique. From this comparison,
the reached depth at 5 kV was estimated to be
around 200 nm. The combination of virtual slic-
ing using BSE DC with physical cutting offers the
possibility of reconstructing very large high-reso-
lution data sets with isotropic voxels.
Figure 2: Point spread function of backscatter
electrons. Image: FEI Co.
nique can create 3-D models of the region 100
to 200 nm below the surface with isotropic
resolution as good as 4 nm throughout. When
combined with serial sectioning, it breaks the
dependence of z-resolution on section thickness, permitting faster, higher-resolution composite models of larger volumes from fewer,
thicker physical sections.
The method also allows for the collection
of high-quality isotropic data, even when the
specimen does not allow the physical removal
of thin enough layers.
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