Mapping the complex morphology of cell interactions with nanowire substrates using FIB-SEM

Abstract

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells' interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered.

Figure 1. Side views of the non-tilted milling obtained image stack of a cell on glass showing the sequential processing operations’ effects.

A) The individual slices have been aligned forming a fairly smooth image using stack-reg algorithm. B) Then the substrate is corrected such as to annul the effects of automatic E-beam shifts in the Slice and View program, resulting in a 52 degree substrate. C) Finally the image stack is rotated 52 degrees to represent the sample on the flat substrate having been cut at an angle.

Figure 2. FIB-SEM image of cell on glass showing front view and top views. A)

Front view shows a non-processed as-imaged slice of a cell on a glass substrate. One can see the nucleus, microvillius, and organelles such as mitochondria in the cell cytosol. The triangular arrows highlight the discrete points where the substrate and cell are in contact. Dashed white lines indicate the two height levels of the horizontal top view sections shown below. H1) Horizontal top view section of the cell close to the substrate level for the fully corrected stack, here it is seen that the cell contacts with the substrate in lines. The two white arrows show one such site where the cell touches the substrate. H2) A top view of the stack is shown higher up in the cell.

Figure 3. Map of the various cell-nanowire interactions observed.

6 cases are outlined with a schematic view and two supporting FIB-SEM images illustrating the case. Case VII, vacuolisation is to a large degree observed in images displaying Case III and Case VI.Inverted view can be found in Figure S3. The close-up images are either regions from the lower magnification image or higher resolution images from a different image.

Figure 4. FIB-SEM image of cells on Nanograss A, illustrating different cell behaviours on the same substrate. A)

FIB-SEM image showing a cell having engulfed broken-off nanowires, and clearly bent silicon nanowires underneath the cell. The nanowires are closely packed in tightly formed clusters inside what appears to be vesicles. B) Another cell on the same substrate, this time the nanowires have been bent by the cell but not completely flattened.

Figure 5. FIB-SEM images of a cell on Nanograss B. A)

As imaged (y-corrected) slice showing the nanowires which appear as white dots due to insufficient sampling frequency. Also worth noting is the example of Case III behaviour with microvili probing the nanograss as outline by the white frame. B) The fully corrected stack can be seen, here the stack has been fully corrected such that independent white dots representing a single nanowire align, illustrating the suboptimal sampling frequency. In some cases the nanowires are not shown from top to bottom as they are slightly tilted compared to the section, quite possibly due to interaction with the cell. Also seen is how the nucleus is avoiding the nanowires (white arrows), and the rippling artefacts which occurs in the corrected front view as previously mentioned.

Figure 6. Image series showing the top view FIB-SEM image of the same cell as in Figure 5 on Nanograss B.

The sections have been made from 5 µm above the substrate to 0.25 µm above the substrate. This illustrates the major forces in play, clearly showing how several nanowires where bent underneath the cell leaving only a few left to indent the nucleus membrane.