An imaging instrument beyond the state of the art
Understanding cellular function requires the characterisation of its molecular constituents as well as their spatial and temporal interaction. To facilitate this, light and atomic force microscopy (AFM) techniques work in a complementary manner to study living cells. While light microscopy allows the visualisation of the interior of cells, AFM provides resolution at the nanometre scale allowing the study of mechanical interactions of membrane components. Optical tweezers (OTs) further enable the observation of molecular interactions inside the cell. The SMW (Single molecule workstation) project aimed to integrate all conceivable functions of these complementary technologies into a single and unified SMW concept. The proposed instrumentation consisted of inverted light microscope (ILM), AFM and OT technologies. By combining these ultra-sensitive microscopy techniques into a single platform, partners aimed to open up completely new horizons for molecular biology-related studies. The setup allowed for simultaneous surface topography using high-resolution AFM, study of the distribution of cellular molecules with enhanced ILM, and measurement of molecular interaction forces with ultra-sensitive OT. As a complementary method, photo-thermal nanospectroscopy was used to investigate spectroscopic properties of cellular material with a high spatial resolution, enabling chemical analysis of sub-cellular structures. The workstation was designed to integrate AFM and ILM from a mechanical, optical and electronic perspective. Additionally, they developed software for these combined technologies coupled with a user interface. This integrated all functional modules of the system, exchanging and controlling measurement protocols in real time and in highly synchronised fashion. Project partners generated a prototype of a combined AFM-ILM instrument and tested its applicability for cancer detection and immunological T cell activation. Optimisation of the SMW platform offered the unique opportunity to simultaneously study the interior and exterior of cells with high resolution. The application of this SMW instrument beyond the project’s lifetime will enable major advances in life science research by delivering new insights into the molecular biology of living cells and complex biomolecular processes, which are currently inaccessible.
Keywords
Cells, atomic force microscopy, SMW, inverted light microscope, molecular biology
