Imaging via widefield surface plasmon resonance microscope for studying bone cell interactions with micropatterned ECM proteins.

The widefield surface plasmon resonance microscope has recently been used to monitor label free antibody/antigen binding events and focal contacts in HaCaT cells at high special resolutions. Thus the aim of this study was to examine MG63 bone cell attachment and alignment to microcontact printed extracellular matrix proteins. Collagen, fibronectin and laminin were stamp patterned onto glass slides using templates consisting of 5-, 10-, 25-, 50- and 100-µm-wide repeat grating. MG63 bone cells were seeded at 50,000 cells per 25 cm(2) and cell alignment was determined from micrographs taken at time-points 2, 5 and 18 h after cell seeding. Cells on the fibronectin pattern attached and elongated at early stages after seeding. In the case of collagen and laminin, cells did not adhere readily and appeared more rounded until 18 h after seeding. This indicated MG63 cells attach mostly via fibronectin specific integrins. The cells aligned well on the fibronectin-patterned cover slips especially to the 50- and 100-µm-wide patterns, although in this case cells did not position themselves in the middle of each fibronectin-coated region, but instead aligned to the small features associated with the edges of the fibronectin-coated regions. Patterned and un-patterned cells also had quite different morphologies. The un-patterned cells had a more rounded morphology and lengths of 25 to 35 µm, whereas patterned cells elongated in the direction of the pattern and had lengths of 50-70 µm. The widefield surface plasmon resonance imaging indicated that cells on un-patterned surfaces had a rounded morphology in which the focal contacts were evenly distributed around the periphery of the cell. However, MG63 bone cells on fibronectin-patterned substrates organized most of their focal contacts along the periphery of the cell distal to the edge of the fibronectin patterns. This suggests that the interaction between the cell and the edge of the pattern induces a reorganization of focal contacts such that the region of the cell guided by the edge of the fibronectin pattern is relatively loosely coupled to the cell culture substrate, but the region of the cell positioned away from that edge is quite tightly coupled to the fibronectin-coated region of the culture substrate. This in turn suggests that guidance is not necessarily associated with enhanced cell substrate coupling along the guidance cue, but may be more associated with a decreased coupling at the guidance cue. Such an arrangement may influence cytoplasmic streaming and as such modulate cell extension. Verification of this finding is required; as such a response to a guidance cue is quite unexpected because it is believed that cells cluster their focal contacts along a guidance cue.

Imaging of the cell surface interface using objective coupled widefield surface plasmon microscopy.

We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.

Development of a novel liquid crystal based cell traction force transducer system.

Keratinocyte traction forces play a crucial role in wound healing. The aim of this study was to develop a novel cell traction force (CTF) transducer system based on cholesteryl ester liquid crystals (LC). Keratinocytes cultured on LC induced linear and isolated deformation lines in the LC surface. As suggested by the fluorescence staining, the deformation lines appeared to correlate with the forces generated by the contraction of circumferential actin filaments which were transmitted to the LC surface via the focal adhesions. Due to the linear viscoelastic behavior of the LC, Hooke's equation was used to quantify the CTFs by associating Young's modulus of LC to the cell induced stresses and biaxial strain in forming the LC deformation. Young's modulus of the LC was profiled by using spherical indentation and determined at approximately 87.1±17.2kPa. A new technique involving cytochalasin-B treatment was used to disrupt the intracellular force generating actin fibers, and consequently the biaxial strain in the LC induced by the cells was determined. Due to the improved sensitivity and spatial resolution (∼1µm) of the LC based CTF transducer, a wide range of CTFs was determined (10-120nN). These were found to be linearly proportional to the length of the deformations. The linear relationship of CTF-deformations was then applied in a bespoke CTF mapping software to estimate CTFs and to map CTF fields. The generated CTF map highlighted distinct distributions and different magnitude of CTFs were revealed for polarized and non-polarized keratinocytes.