High-resolution imaging using a novel atomic force microscope and confocal laser scanning microscope hybrid instrument: essential sample preparation aspects.

The recent data explosion in global gene expression profiling and proteomics has resulted in a need to determine the mechanistic role of biomarker signatures in pathogenicity. Consequently, elaborate technologies are required to assess increasingly smaller sub-cellular compartments and constituents. We describe the development, evaluation and application of an efficient sample preparation methodology to facilitate coupled atomic force microscopy and confocal laser scanning microscopy (AFM-CLSM), providing a novel means of concurrent high-resolution structural and fluorescence imaging. Due to their fragile nature and nanoscale dimensions, filopodia were selected as a model to develop the procedure that maximised fluorescence response, while maintaining epithelial cell ultra-structure. Fixation with ultra-pure methanol-free formaldehyde coupled to quantum dot nanocrystal labelling proved to be vital in achieving high quality AFM-CLSM images. We demonstrated for the first time that filopodia have a "quilted" surface structure. Additionally, high ultra-structural ridges on the apical cell surface resolved by AFM corresponded to punctate moesin clusters, representing direct visualisation of moesin linkages between transmembrane proteins and the cytoskeleton. The capacity of this novel multi-modal imaging technique to probe topography, molecular composition and biophysical properties of ultra-structural features therefore provides unique information that will significantly contribute to our understanding of cellular structure-function relationships.