Thursday 27.March 2014, 13.45 - 14.30
Bridging Microscopes: 3D correlative light and scanning electron
microscopy of complex biological structures
MS Lucas, M Günthert, F Lucas, P Gasser, Anne-Greet Bitterman, Chris Buser and R
Wepf (Zuerich, S)
Correlative light and electron microscopy (CLEM) is a powerful tool in life science to
combine large-scale volume imaging of cells or tissues by LM with a high-resolution
description of their morphology using EM. The combination of 3D microscopy
techniques such as CLSM with FIB-SEM or serial block face SEM (SBF-SEM) [1] or
array tomography (AT) [2] opens up new possibilities to expand morphological
context description and analysis into to the third dimension on the nm-scale.
These three approaches to correlate 3D LM data with SEM volume data have been
shown to provide a valid alternative to TEM-based serial sectioning approaches [3].
Modern SEM-platforms allow imaging with an x/y-resolution of 2-3 nm, and offer the
advantage of automation: e.g. routine overnight FIB-SEM volume imaging can cover
cross-sections of up to 40×20 µm2. With a slice thickness of 5-10 nm, these stacks
can expand to several 10 µm in the z-axis. FIB-SEM further enables precise, sitespecific localisation and milling.
SBF-SEM offers more flexibility when choosing the volume size: theoretically the area
to be scanned is limited only by the size of the block face. However, very large scan
areas come at the cost of the x/y pixel size or extended scan time (BSE imaging). The
slice thickness can be chosen from 15 - 200 nm to ideally match the biological
question. Thus areas from several 100x100µm and several 100µm in depth (volumes:
10000 to 6*106 µm³) can be recorded [4].
In combination with prior screening of the specimen by confocal LM to identify the
ROI, this allows targeted high-resolution 3D imaging of the structure of interest. This
approach requires a sample preparation that facilitates the application of both LM
and EM on a bulk specimen. To make this possible we have adapted protocols for
freeze-substitution after high-pressure freezing, or chemical fixation and embedding
in resin to include fluorophores in the samples [5].
Thus fluorescently labeled, resin-embedded specimens can also be used for AT. Here,
ribbons of serial sections are loaded onto glass-slides, enabling even larger ROIs to be
investigated by wide-field LM and subsequently by SEM. With the introduction of
special surface coatings such as ITO these glass supports remain transparent for LM,
but become highly conductive for SEM investigations. Such ribbons can be stained for
histology (e.g. Toluidine blue or H&E) or for fluorescent immuno-labeling to
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specifically identify a ROI by F/LM prior to HR-SEM imaging. If needed, the sections
can also be post-stained with uranyl acetate for (FIB)SEM-imaging.
Software solutions are available to facilitate the relocation of the ROI by a markerbased calibration of the sample in both LM and (FIB-)SEM. And automated recording
of 3D volumes is possible for all three described methods. However, the FIB-SEM
approach can achieve the better z-resolution, whereas the z-resolution for AT and
SBF-SEM is limited by the sectioning process. FIB-SEM can therefore record isotropic
voxels, which is an advantage when it comes to 3D reconstruction and modeling.
SBF-SEM and AT on the other hand provide a much larger field of view. And AT even
enables specific fluorescence labelling to identify the ROI combined with immunegold labelling for HR-SEM. For SBF-SEM the ROI has to be defined using the
information which can be obtained from the surface of the block face, either by LM
or SEM. Furthermore, FIB-SEM and SBF-SEM are destructive methods, while the ATribbons can be stored and re-investigated ad libitum [6].
Resulting 3D datasets from LM and EM imaging have very different resolutions but
can be merged in-silico to 3D models. By combining with other imaging modalities
(e.g Xray-CT) one can reconstruct the structural hierarchical scaffold and hence
better understand the complexity of living systems. Further transforming these
structures into a visual perception space or haptic perception space by 3D printing
the nano-world becomes better (comprehensive) accessible also for the layman. The
method of choice has to be determined anew for every project, according the sample
type, the size of the structure or the volume of interest and the desired imaging
resolution in x,y, and z.
References
[1] W Denk et al, PLoS Biol 2 (2004), p. e329.; [2] KD Micheva et al, Neuron 55 (2007), p. 25.
[3] MS Lucas et al, Methods Mol. Biol. 1117 (2014), p. 593.;[4] CJ Peddie et al, Micron (2014), p. epub
ahead of print.; [5] SS Biel et al, J. Microsc. 212 (2003), p. 91.; [6] MS Lucas et al, Methods Cell Biol. 111
(2012), p. 325.
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