least threefold to the micron range. Both techniques can be used on plastic sections
or cryosections, but the latter avoids all the pitfalls and artefacts of chemical fixation. However, cryo-ultramicrotomy is not for the faint hearted and remains the
“Holy Grail” for most of us. It also introduces a major artefact in the compression of
sections and formation of crevasses. Yet new developments in the last 5 years will
make this an artefact from the past with the preparation of focused ion beam
cryo-lamellas instead of the cryosections. Though this is, for now, still only applied
in a few laboratories that have the resources and more importantly the rare skill sets
required. Dual beam microscopes have also seen an increased usage in biological
sciences with the introduction of serial block face imaging where the microscope
sequentially section and image the surface of the sample therefore creating a
three-dimensional dataset at intermediate resolution between optical and transmission electron microscopy. The same techniques can be applied using a microtome
inside the scanning electron microscope chamber creating virtual sections. Indeed
the sections are actually accumulating on the surface of the knife and cannot be
recovered.
So we have now moved full circle and are back to serial sections using the
slightly less complicated tool that is the scanning electron microscope. Will this
practice be the future of electron microscopy for the masses?
For now, all these techniques are complementary and one cannot replace fully
the others. While plastic electron tomography is often seen as the poor parent of
cryo-electron tomography, it is indisputable that the output in terms of number of
samples per unit of time is far greater. In turn, cryo-ultramicrotomy is far faster than
lamella preparation using focused ion beams, while block face imaging is for large
samples at slightly lower resolution.
At the fringe of these three-dimensional techniques are applications that link
them to the world of fluorescence microscopy and structural biology. On one side is
array tomography, still using serial sections but with fluorescently labelled probes
that let the scientist identify the otherwise grey blob. Or what used to be blobs, as
subtomogram averaging is progressing fast, thanks to the direct electron detectors.
This permits to do structural biology at a resolution not quite as high as single
particle, but still below the 10 Å mark. The main advantage of this technique is to
be able to place the structure in the context of the whole cell.
Furthermore, the acquisition of images is one thing, but the annotation and
segmentation require some additional processing in order to be able to handle data
with low signal-to-noise ratio (e.g. cryo-electron tomography).
Finally, the propagation of these techniques, their automation, and therefore the
amount of data generated, have led to what are probably some of the biggest
challenges the field has encountered and will have to face for the next few years:
data storage and management. With these techniques going towards full automation
some skills are likely to be lost, i.e. with the introduction of kits in molecular
biology how many students still know what diatomaceous earth looks likes and
vi
Preface
or cryosections, but the latter avoids all the pitfalls and artefacts of chemical fixation. However, cryo-ultramicrotomy is not for the faint hearted and remains the
“Holy Grail” for most of us. It also introduces a major artefact in the compression of
sections and formation of crevasses. Yet new developments in the last 5 years will
make this an artefact from the past with the preparation of focused ion beam
cryo-lamellas instead of the cryosections. Though this is, for now, still only applied
in a few laboratories that have the resources and more importantly the rare skill sets
required. Dual beam microscopes have also seen an increased usage in biological
sciences with the introduction of serial block face imaging where the microscope
sequentially section and image the surface of the sample therefore creating a
three-dimensional dataset at intermediate resolution between optical and transmission electron microscopy. The same techniques can be applied using a microtome
inside the scanning electron microscope chamber creating virtual sections. Indeed
the sections are actually accumulating on the surface of the knife and cannot be
recovered.
So we have now moved full circle and are back to serial sections using the
slightly less complicated tool that is the scanning electron microscope. Will this
practice be the future of electron microscopy for the masses?
For now, all these techniques are complementary and one cannot replace fully
the others. While plastic electron tomography is often seen as the poor parent of
cryo-electron tomography, it is indisputable that the output in terms of number of
samples per unit of time is far greater. In turn, cryo-ultramicrotomy is far faster than
lamella preparation using focused ion beams, while block face imaging is for large
samples at slightly lower resolution.
At the fringe of these three-dimensional techniques are applications that link
them to the world of fluorescence microscopy and structural biology. On one side is
array tomography, still using serial sections but with fluorescently labelled probes
that let the scientist identify the otherwise grey blob. Or what used to be blobs, as
subtomogram averaging is progressing fast, thanks to the direct electron detectors.
This permits to do structural biology at a resolution not quite as high as single
particle, but still below the 10 Å mark. The main advantage of this technique is to
be able to place the structure in the context of the whole cell.
Furthermore, the acquisition of images is one thing, but the annotation and
segmentation require some additional processing in order to be able to handle data
with low signal-to-noise ratio (e.g. cryo-electron tomography).
Finally, the propagation of these techniques, their automation, and therefore the
amount of data generated, have led to what are probably some of the biggest
challenges the field has encountered and will have to face for the next few years:
data storage and management. With these techniques going towards full automation
some skills are likely to be lost, i.e. with the introduction of kits in molecular
biology how many students still know what diatomaceous earth looks likes and
vi
Preface