91 beams [94], and most recently even 196 beams [95]. These have been developed
to improve image acquisition times by several orders of magnitude and could
revolutionise the speed at which SBEM work could be done. Eberle et al. [96]
demonstrate the potential of the 61 beam to image the block-face of a resin
embedded mouse brain sample.
While volume SEM, particularly SBEM and FIB-SEM, have now made it
possible to achieve true 3D structural data of biological systems in an automated
fashion there is still a need for considerable developments in these techniques.
Advances in speed, resolution and sample preparation are important to make these
techniques more routine. Speed and resolution will be achieved as a result of new
instrumentation, both in microscopes and detectors. The production of a truly
conductive and beam stable resin will revolutionise SBEM as it will relieve the
many problems associated with cutting and imaging that are now inherent with this
technique. Improvements to automated segmentation, which is the stumbling block
of analysing these large data sets will lessen the workload and allow production of
results in a more timely fashion. Volume SEM is technically challenging and so
improvements to the entire workflow will assist in an even wider adoption by
biological researchers.
Acknowledgements Thanks to the staff from both the EMBL Electron Microscopy Core Facility
and the University of Queensland Centre for Microscopy and Microanalysis. The constant support
from Yannick Schwab cannot be surpassed. We are also thankful to Anna Steyer and Rachel
Mellwig for their helpful discussions and suggestions to the manuscript. A special mention to
Matthia Karreman and José Miguel Serra Lleti. Without the work of Robyn Chapman we wouldn’t
have a SBEM system and it’s her untiring efforts that have led to its development in our facilities.
I thank her for all her determination to make this technique a success. Rob Parton has always been
a source of encouragement for this work and has been willing to supply samples for us to work
with when developing new ideas. Rachel Templin has been a wonderful backup helping us to
process large numbers of samples as we have sought to develop new processing for this
technology.
References
1. A. Birch-Andersen, Reconstruction of the nuclear sites of Salmonella typhimurium from
electron micrographs of serial sections. J. Gen. Microbiol. 13(2), 327–329 (1955)
2. B.G. Bang, F.B. Bang, Graphic reconstruction of the third dimension from serial electron
microphotographs. J. Ultrastruct. Res. 1, 138–146 (1957). doi:10.1016/S0022-5320(57)
80002-1
3. J.G. White, E. Southgate, J.N. Thomson, S. Brenner, The structure of the nervous system of
the nematode Caenorhabditis elegans: the mind of a worm. Phil. Trans. R. Soc. London 314,
1–341 (1986)
4. K.L. Briggman, M. Helmstaedter, W. Denk, Wiring specificity in the direction-selectivity
circuit of the retina. Nature 471, 183–188 (2012). doi:10.1038/nature09818
5. W. Denk, H. Horstmann, Serial block-face scanning electron microscopy to reconstruct
three-dimensional tissue nanostructure. PLoS 2(11), e329–10 (2004). doi:10.1371/journal.
pbio.0020329
5 Volume Scanning Electron Microscopy: Serial Block-Face …
143
to improve image acquisition times by several orders of magnitude and could
revolutionise the speed at which SBEM work could be done. Eberle et al. [96]
demonstrate the potential of the 61 beam to image the block-face of a resin
embedded mouse brain sample.
While volume SEM, particularly SBEM and FIB-SEM, have now made it
possible to achieve true 3D structural data of biological systems in an automated
fashion there is still a need for considerable developments in these techniques.
Advances in speed, resolution and sample preparation are important to make these
techniques more routine. Speed and resolution will be achieved as a result of new
instrumentation, both in microscopes and detectors. The production of a truly
conductive and beam stable resin will revolutionise SBEM as it will relieve the
many problems associated with cutting and imaging that are now inherent with this
technique. Improvements to automated segmentation, which is the stumbling block
of analysing these large data sets will lessen the workload and allow production of
results in a more timely fashion. Volume SEM is technically challenging and so
improvements to the entire workflow will assist in an even wider adoption by
biological researchers.
Acknowledgements Thanks to the staff from both the EMBL Electron Microscopy Core Facility
and the University of Queensland Centre for Microscopy and Microanalysis. The constant support
from Yannick Schwab cannot be surpassed. We are also thankful to Anna Steyer and Rachel
Mellwig for their helpful discussions and suggestions to the manuscript. A special mention to
Matthia Karreman and José Miguel Serra Lleti. Without the work of Robyn Chapman we wouldn’t
have a SBEM system and it’s her untiring efforts that have led to its development in our facilities.
I thank her for all her determination to make this technique a success. Rob Parton has always been
a source of encouragement for this work and has been willing to supply samples for us to work
with when developing new ideas. Rachel Templin has been a wonderful backup helping us to
process large numbers of samples as we have sought to develop new processing for this
technology.
References
1. A. Birch-Andersen, Reconstruction of the nuclear sites of Salmonella typhimurium from
electron micrographs of serial sections. J. Gen. Microbiol. 13(2), 327–329 (1955)
2. B.G. Bang, F.B. Bang, Graphic reconstruction of the third dimension from serial electron
microphotographs. J. Ultrastruct. Res. 1, 138–146 (1957). doi:10.1016/S0022-5320(57)
80002-1
3. J.G. White, E. Southgate, J.N. Thomson, S. Brenner, The structure of the nervous system of
the nematode Caenorhabditis elegans: the mind of a worm. Phil. Trans. R. Soc. London 314,
1–341 (1986)
4. K.L. Briggman, M. Helmstaedter, W. Denk, Wiring specificity in the direction-selectivity
circuit of the retina. Nature 471, 183–188 (2012). doi:10.1038/nature09818
5. W. Denk, H. Horstmann, Serial block-face scanning electron microscopy to reconstruct
three-dimensional tissue nanostructure. PLoS 2(11), e329–10 (2004). doi:10.1371/journal.
pbio.0020329
5 Volume Scanning Electron Microscopy: Serial Block-Face …
143