Tissue section AFM: In situ ultrastructural imaging of native biomolecules.
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-microm 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.
Unit of Cardiac Physiology, School of Biomedicine, The University of Manchester, Manchester Academic Health Science Centre, Manchester, M13 9PT, UK.
Helen K
HK
Graham
Nigel W
NW
Hodson
Judith A
JA
Hoyland
Sarah J
SJ
Millward-Sadler
David
D
Garrod
Anthea
A
Scothern
Christopher E M
CE
Griffiths
Rachel E B
RE
Watson
Thomas R
TR
Cox
Janine T
JT
Erler
Andrew W
AW
Trafford
Michael J
MJ
Sherratt
D002356:Cartilage / Q000378:metabolism / Q000648:ultrastructure*; D003952:Diagnostic Imaging / Q000379:methods; D005109:Extracellular Matrix / Q000378:metabolism / Q000648:ultrastructure*; D016591:Histocytological Preparation Techniques / Q000379:methods; D006801:Humans; D008853:Microscopy / Q000379:methods*; D018625:Microscopy, Atomic Force / Q000379:methods*