Thomas R. Cox, May 2010
Our recent collaborative work with the University of Manchester has just been published in Matrix Biology. The paper, titled ‘‘Tissue section AFM: In situ ultrastructural imaging of native biomolecules’ looks at the application of Atomic Force Microscopy (AFM) imaging to understand the organisation of tissues. We show that a combination of cryo-sectioning with non-destructive AFM can be used to preserve and visualise extracellular matrix assemblies at the nanometer scale, and can be used together with conventional microscopy and micro-mechanical profiling techniques.
The approach allows high resolution imaging of freeze dried or critical point dried specimens to maximize structural preservation or samples in physiological buffers. The latter approach will enable the quantitation of dynamic changes in tissue structures due to in vitro assembly or degradation and may be important in deepening our understanding of extracellular matrix remodelling.
Conventional approaches for ultrastructural high-resolution imaging of biological specimens induce profound changes in bio-molecular structures. By combining tissue cryo-sectioning with non-destructive atomic force microscopy (AFM) imaging we have developed a methodology that may be applied by the non-specialist to both preserve and visualize bio-molecular structures (in particular extracellular matrix assemblies) in situ. This tissue section AFM technique is capable of: i) resolving nm–µm scale features of intra- and extracellular structures in tissue cryo-sections; ii) imaging the same tissue region before and after experimental interventions; iii) combining ultrastructural imaging with complimentary microscopical and micromechanical methods. Here, we employ this technique to: i) visualize the macro-molecular structures of unstained and unfixed fibrillar collagens (in skin, cartilage and intervertebral disc), elastic fibres (in aorta and lung), desmosomes (in nasal epithelium) and mitochondria (in heart); ii) quantify the ultrastructural effects of sequential collagenase digestion on a single elastic fibre; iii) correlate optical (auto fluorescent) with ultrastructural (AFM) images of aortic elastic lamellae.
Graham HK, Hodson NW, Hoyland JA, Millward-Sadler SJ, Garrod D, Scothern A, Griffiths CE, Watson RE, Cox TR, Erler JT, Trafford AW, Sherratt MJ. Tissue section AFM: In situ ultrastructural imaging of native biomolecules
Matrix Biology; May;29(4):254-60 (2010) | doi: 10.1016/j.matbio.2010.01.008.
This work was supported by Research into Ageing, the British Heart Foundation, by a SPARC (Strategic Promotion of Ageing Research Capacity) award, the Medical Research Council and the NIHR Manchester Biomedical Research Centre.