title: Mechanisms of cell-cell adhesion identified by immunofluorescent labelling with quantum dots: a scanning near-field optical microscopy approach.
abstract: Scanning near-field optical microscopy (SNOM) has been employed to simultaneously acquire high-resolution fluorescence images along with shear-force atomic force microscopy from cell membranes. Implementing such a technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The application of the technique to investigate cell-cell adhesion has revealed the interactions of filopodia and their functional relationship in establishing adherens junctions. This has been achieved via the selective tagging of the cell adhesion protein, E-cadherin, by immunofluorescence labelling. Two labelling routes were explored; Alexa Fluor 488 and semiconductor quantum dots. The quantum dots demonstrated significantly enhanced photostability and high quantum yield making them a versatile alternative to the conventional organic fluorophores often used in such a study. Analysis of individual cells revealed that E-cadherin is predominantly located along the cell periphery but is also found to extend throughout their filopodia. We have demonstrated that with a fully optimised sample preparation methodology, quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.
authors.affiliation: School of Physical Sciences, Department of Physics, Multidisciplinary Nanotechnology Centre, Swansea University, Singleton Park, Swansea SA2 8PP, UK.
authors.forename: K-A D
authors.initials: KA
authors.lastname: Walker
authors.forename: S H
authors.initials: SH
authors.lastname: Doak
authors.forename: P R
authors.initials: PR
authors.lastname: Dunstan
mesh_terms: D015820:Cadherins / Q000502:physiology; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods*; D011554:Pseudopodia / Q000502:physiology; D045663:Quantum Dots
chemical_list: D015820:Cadherins; D005456:Fluorescent Dyes