Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues
Background: Imaging large volumes such as entire cells or small model organisms at nanoscale resolution seemed
an unrealistic, rather tedious task so far. Now, technical advances have lead to several electron microscopy (EM)
large volume imaging techniques. One is array tomography, where ribbons of ultrathin serial sections are deposited
on solid substrates like silicon wafers or glass coverslips.
Results: To ensure reliable retrieval of multiple ribbons from the boat of a diamond knife we introduce a substrate
holder with 7 axes of translation or rotation specifically designed for that purpose. With this device we are able to
deposit hundreds of sections in an ordered way in an area of 22 × 22 mm, the size of a coverslip. Imaging such
arrays in a standard wide field fluorescence microscope produces reconstructions with 200 nm lateral resolution
and 100 nm (the section thickness) resolution in z.
By hierarchical imaging cascades in the scanning electron microscope (SEM), using a new software platform, we
can address volumes from single cells to complete organs. In our first example, a cell population isolated from
zebrafish spleen, we characterize different cell types according to their organelle inventory by segmenting 3D
reconstructions of complete cells imaged with nanoscale resolution. In addition, by screening large numbers of
cells at decreased resolution we can define the percentage at which different cell types are present in our
preparation. With the second example, the root tip of cress, we illustrate how combining information from
intermediate resolution data with high resolution data from selected regions of interest can drastically reduce the
amount of data that has to be recorded. By imaging only the interesting parts of a sample considerably less data
need to be stored, handled and eventually analysed.
Conclusions: Our custom-designed substrate holder allows reproducible generation of section libraries, which can
then be imaged in a hierarchical way. We demonstrate, that EM volume data at different levels of resolution can
yield comprehensive information, including statistics, morphology and organization of cells and tissue. We predict,
that hierarchical imaging will be a first step in tackling the big data issue inevitably connected with volume EM.
|Zugehörige Institution(en) am KIT
||Institut für Angewandte Informatik (IAI)
KITopen ID: 1000065550
||43.22.03; LK 01
||BMC cell biology
||Array tomography, Serial sectioning, Section libraries, Hierarchical imaging, Large volume 3D reconstruction
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