Ultra‐thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures

Abstract

The preparation of biological cells for either scanning or transmission electron microscopy requires a complex process of fixation, dehydration and drying. Critical point drying is commonly used for samples investigated with a scanning electron beam, whereas resin-infiltration is typically used for transmission electron microscopy. Critical point drying may cause cracks at the cellular surface and a sponge-like morphology of nondistinguishable intracellular compartments. Resin-infiltrated biological samples result in a solid block of resin, which can be further processed by mechanical sectioning, however that does not allow a top view examination of small cell-cell and cell-surface contacts. Here, we propose a method for removing resin excess on biological samples before effective polymerization. In this way the cells result to be embedded in an ultra-thin layer of epoxy resin. This novel method highlights in contrast to standard methods the imaging of individual cells not only on nanostructured planar surfaces but also on topologically challenging substrates with high aspect ratio three-dimensional features by scanning electron microscopy.

Cellular interfaces with hydrogen-bonded organic semiconductor hierarchical nanocrystals

“…4). The evaluation of the true interface cleft distance in vivo is a question of current debate, as the fixation of cells for electron microscopy can produce a cleft morphology different from what is present in vivo 15, 54 . The cleft distance has been determined to change as much as 10–50 nm following fixation in some cases, even where cryofixation methods are employed.…”

How to image cell adhesion on soft polymers?

“…To preserve cells for SEM, they were embedded in epoxy-resin by stepping from pure ethanol to pure resin as described previously. 11 2.5 FIB cuts-The samples were glued to a SEM stub using silver glue (Plano GmbH). To protect the cells from the ion and electron beams the samples were sputtered for 30s at 15mA with platinum (a 1-1.5 µm thick layer).…”

Revealing the Cell–Material Interface with Nanometer Resolution by Focused Ion Beam/Scanning Electron Microscopy

“…However, using FIB-SEM to examine the cell-to-materials interface is severely limited by the lack of contrast of biological specimens and the sponge-like intracellular defects induced by hard drying procedures 22–24 . Resin embedding preparation with heavy metals allow the visualization of intracellular structures even in proximity of nanostructures 25,26 but the resin matrix around the cells does not allow any visualization of the entire cells unless a 3D reconstruction of the whole specimen is performed.…”

“…In our method, after resin infiltration and before resin polymerization, a resin-removal step is introduced that strips off the excess extracellular resin by first draining and, then, flushing the sample with ethanol. This step thins down the resin coating outside the cell membrane to tens of nanometers while maintaining a stable intracellular resin embedding 24 . The final step involves curing the liquid resin to a thin layer of plastic with cells embedded inside.…”