Optimisation of sample preparation methods for air imaging of ocular mucins by AFM

• Published in: Ultramicroscopy 2003-10, Vol.97 (1), p.289-296
• Main Authors: Brayshaw, Debra J., Berry, Monica, McMaster, Terence J.
• Format: Article
• Language: English
• Subjects: Air; Aluminum Silicates; Atomic force microscopes; Biological and medical sciences; Buffers; Cation; Conjunctiva - chemistry; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; General aspects, investigation technics, apparatus; Glycoprotein; Humans; Instruments, apparatus, components and techniques common to several branches of physics and astronomy; Microscopy, Atomic Force - methods; Molecular resolution; Mucins - ultrastructure; Nickel; Physics; Physisorption; Scanning probe microscopes, components and techniques; Scanning tunneling and atomic force microscopy; Specimen Handling - methods; Tissues, organs and organisms biophysics
• ISSN: 0304-3991; 1879-2723
• DOI: 10.1016/S0304-3991(03)00054-8

The addition of cations to the imaging buffer for AFM has been previously shown to improve the binding of biological molecules to mica. Investigations were carried out to find the concentration of NiCl 2 required to immobilize mucin molecules on a freshly cleaved mica surface, for imaging using intermittent contact in air. Drop-deposition of samples prepared in HEPES buffer with 1, 2 and 5 mM NiCl 2 revealed the sensitivity of the mucin molecules to salt. Dialysis of the mucin solutions dramatically reduced the amount of salt present and allowed single molecules to be imaged, revealing a variation in thickness along their length. Spray deposition of the same mucin solutions produced single molecules that, although less affected by co-adsorbed salt, showed a degree of self-folding. This shows the sensitive balance between HEPES and NiCl 2 required for successful imaging of the sub-molecular features of individual mucin molecules.