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

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Abstract

Summary

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.

Lay Description

For decades, biological materials like cells have been analyzed with optical microscopy. While this powerful tool for the investigation of cells reaches down to the sub-micron regime, it remains limited to visualize the finest cell compartments and organelles in the very few nanometer regime. This limitation can be overcome by the use of electron microscopy, which can reach extremely highly resolution to satisfy that requirement. The artifact-free transition of living cells into a solid state in order to be inspected in an ultrahigh vacuum remains a challenge. That procedure consists typically of three steps. Firstly, cells need to be chemically fixed and, thereby, morphologically stabilized. Secondly, they need to be dehydrated in order to exchange the water which is typically achieved by an organic solvent. The final crucial step prepares the dehydrated cells in such a way that cells remain in a solid state. Critical point drying (CPD) is a commonly chosen technique as a drying method, in particular for scanning electron microscopy (SEM). This method is based on the thermodynamical pathway via a supercritical liquid. For transmission electron microscopy an alternative method was developed, using resin-infiltration of an organic substance into the cell. While CPD typically might cause cracks at the cellular surface, resin remains as a solid block of material also on top of the cells. The infiltration of the material stabilizes the inner organelles by not leaving empty areas inside of the cell behind. Subsequently, the cell suffers less structural damages as in the case of the CPD. Finally, we introduced a method which removes resin excess on the sample before the resin is fully polymerized. This method allows to gain access to the surface morphology of the cell which previously could not be inspected with scanning electron microscopy but only with CPD. We applied this method for the imaging of single cells on planar and 3D nanostructured surfaces. We show in addition that this method is excellently suited for sequential cross section with a focused ion beam and, eventually, visualizing the intracellular ultrastructure to a very high degree.

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