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pagetitle-crest Dual Probe Atomic Force Microscopy

pagetitle-crest Dual probe AFM instrumentation and multilevers

DJ Brayshaw & TJ McMaster

Collaborators: P Heard (Interface Analysis Centre)

Atomic Force Microscopy (AFM) has catalysed intensive research into biological and single molecule experiments. We can routinely immobilise and obtain ultra-high resolution topographic images of single long-chain molecules, often in an hydrated state. From the images we can glean structural information, and occasionally observe a slow timescale process. What is lacking in this direct imaging technique is the ability to measure, or map, the variation in functionality and properties across a surface and to directly correlate this with the topographical information.

To address this current limitation, we have carried out investigations to fabricate and characterise novel AFM multi-cantilevers whose spatial arrangement and tip geometry will be controlled using focused ion beam (FIB) techniques. By varying the multi-probe designs significant progress has been made towards separating torsional and flexural probe responses. Designs have been produced with the aim of fabricating and developing multi-functional AFM probes capable of simultaneously probing multiple chemical and physical properties of the same molecule.

dual_afm1

Conventional AFM image of dehydrated ocular mucins.

dual_afm2

Dual-beam AFM cantilevers modified by FIB.

 dual_afm3

Distinct resonant frequencies vs dual laser AFM head.

Besides controlling the specific geometries of the cantilevers, concurrent work is focussing on the development of a dual laser diode AFM head for individual, unambiguous monitoring of the two cantilever signals. Alternatively, it is possible to design and fabricate cantilevers where each beam has a unique resonant frequency allowing the signals can be isolated accordingly.


pagetitle-crest Dual probe AFM & ocular mucins

DJ Brayshaw, SC Baos & TJ McMaster

Collaborators: M Berry (Ophthalmology)

Ocular mucins represent the primary sample to be investigated using the dual probe AFM. Mucins are a family of large glycoproteins and therefore have the generic macromolecular structure of a thread-like peptide backbone decorated by densely packed carbohydrate side chains. Ocular mucins are complex glycoconjugates, secreted by all cells of the ocular epithelium, including specialist mucin secreting cells, (goblet cells), in the conjunctiva.

dual_muc1

750 x 750 nm image of purified dehydrated ocular mucin on mica. Variation in thickness is clearly visible along the backbone of the molecule.

 dual_muc2

Schematic representation of secreted, gel forming mucins highlighting disulphide-linked peptide core and dense oligosaccharide side chains.

Secreted mucins typically form large polymers through linkage of subunits via disulphide bonds. One possible use for the dual probe AFM is local deposition of a disulphide bond breaking agent to controllably reduce the polymer chains to their subunits.

Ocular mucins in their native environment are integrated in the pre-ocular fluid in a gel-like state. Gels present possibly the ultimate challenge to AFM imaging, in which a mechanical interaction with the sample is required. By using the dual probe AFM as a simultaneous imaging and force spectroscopy tool (one functionalised and one unmodified tip), it is hoped that new structural information can be gleaned whilst ensuring that the integrity of the gel-like structure is not damaged.

More information on conventional AFM imaging of mucins can be found here.

dual_muc3

250 x 250 nm image of the macromolecular, artificial gel assembly of purified mucins. The molecules appear entangled, densely but irregularly spaced, yielding an image qualitatively similar to that of an agarose gel.