What's Happening on the Other Side? Revealing Nano-Meter Scale Features of Mammalian Cells on Engineered Textured Tantalum Surfaces

Abstract

Advanced engineered surfaces can be used to direct cell behavior. These behaviors are typically characterized using either optical, atomic force, confocal, or electron microscopy; however, most microscopic techniques are generally restricted to observing what's happening on the "top" side or even the interior of the cell. Our group has focused on engineered surfaces typically reserved for microelectronics as potential surfaces to control cell behavior. These devices allow the exploration of novel substrates including titanium, tungsten, and tantalum intermixed with silicon oxide. Furthermore, these devices allow the exploration of the intricate patterning of surface materials and surface geometries i.e., trenches. Here we present two important advancements in our research: (1) the ability to split a fixed cell through the nucleus using an inexpensive three-point bend micro-cleaving technique and image 3D nanometer scale cellular components using high-resolution scanning electron microscopy; and (2) the observation of nanometer projections from the underbelly of a cell as it sits on top of patterned trenches on our devices. This application of a 3-point cleaving technique to visualize the underbelly of the cell is allowing a new understanding of how cells descend into surface cavities and is providing a new insight on cell migration mechanisms.

Keywords: adhesion; cross-sectioning; mammalian cells; morphology; nanoscale; tantalum.

Figure 1

(a) Schematic drawing of the three-point bend micro-cleaving. (b) Cell dissection obtained using three-point bend technique. Substrate is covered with ~20 nm tantalum film (green).

Figure 2

Fluorescence confocal micrographs show cell nuclei (blue) and actin micro-filaments (red) randomly oriented on blanket tantalum surfaces.

Figure 3

Low- and high-magnification fluorescence confocal micrographs of cells adhered to 0.18 μm (a–d), 0.5 μm (e–h), and 2 μm comb structures (i–l). The nuclei (blue), actin microfilaments (red) aligned to the pattern axes. Cells were incubated for 24 h with a concentration of ~5 × 10⁵ cells/mL.

Figure 4

Typical 70° tilted low-magnification SEM micrographs of cells on comb structures with trench widths of (a) 0.18 μm and (b) 0.25 μm. Note that cellular material within the trenches is not visible. A cross-sectioning technique is needed to visualize the underbelly of the cell.

Figure 5

Tilted SEM micrographs of two cross-sectioned cells. (a–b) Cell adhered on two types of surface (flat and 0.18 μm trench pattern). (c–e) High magnification images of the fractured surface in (b). (f–g) Cell adhered solely on a flat surface. Both sides of the cleaved surfaces are displayed in the figure (left and right columns of images). Landmarks “1–“4” are used to match the topographic features on the two fractured surfaces. In both cells, a micron size sub-nuclear organelle was cross-sectioned.

Figure 6

Typical SEM micrographs of cross-sectioned cells on the comb structures with trench widths of (a) 0.18, (b) 0.25, (c) 0.5, (d) 2, and (e) 50 μm.

Figure 7

Schematic drawings of three different types of cell cross-sectional morphology observed with increasing line and trench widths. (a) Cell “floated” on top of the comb structure; (b) pseudopodia located at the edge of cells descended into the trenches; (c) entire cell including the nucleus formed a conformal coating on the comb structure surface.

Figure 8

Micrographs of dissected cells show different amounts of cellular material descending into the 2 μm trenches. The size of filament cluster or fibrous structures that connect the cell underbelly to the substrate increased as more materials sunk into the trench. (a) Short filaments protruding out of the cell underbelly but that did not attach to any surface; (b-c) single-strand filaments from the cell underbelly attached to the bottom of trenches; (d) a cluster of filaments connected the cell underbelly and the bottom of trenches; (e) a large network of fibrous structures is formed between the cell and the trench bottom surfaces.

Figure 9

Micrographs show cell adhered to the 2 μm comb structures. (a) The sub-nuclear organelle resting on the line is shown in (a) while the organelle sat in the trench is shown in (b).