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.
Highlights
Nano- and micro-fabricated structured substrates achieve an increasing amount of interest in cell biology, where their uses are as diverse as biochemical manipulation [1,2], supporting and controlling cell movement [3,4,5], electrophysiological measurements [6,7,8] and intracellular measurements [9,10]
In this paper we present a study of block face focused ion beam scanning electron microscope (SEM) (FIB-SEM) imaging of polymer embedded 3T3 Fibroblast cell monolayers on nanostructured substrates without prior removal of the substrate
Cells on a Flat Substrate For the NIH3T3 cells cultured on the unstructured blank sample of flat Pyrex glass and investigated with focused ion beam (FIB)-SEM the final stack’s resolution given by the pixels of the original image was 10 nm in X direction, 10 nm in Y direction and 100 nm in Z direction
Summary
Nano- and micro-fabricated structured substrates achieve an increasing amount of interest in cell biology, where their uses are as diverse as biochemical manipulation [1,2], supporting and controlling cell movement [3,4,5], electrophysiological measurements [6,7,8] and intracellular measurements [9,10]. Examples in literature often show images of critically point dried (CPD) cells imaged by a scanning electron microscope (SEM) This method provides excellent images showing how cells lie on the particular substrate, and one can get an idea of the level of interaction with the substrate by cell protrusions such as lamellipodia [2,4,10,13,14].
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