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

Histochemistry and Cell Biology
Shareen H DoakChris Wright

Abstract

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 prot...Continue Reading

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Citations

Dec 1, 2009·Histochemistry and Cell Biology·Wolfgang-Moritz Heupel, Detlev Drenckhahn
May 9, 2014·Expert Opinion on Drug Delivery·Constanze LamprechtAndreas Ebner
Aug 6, 2010·Brain Research Reviews·Filipa Lourenço CardosoMaria Alexandra Brito
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Jun 15, 2010·Analytical Chemistry·Aleeta M PoweIsiah M Warner

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