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Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells.

Miragoli M, Moshkov A, Novak P, Shevchuk A, Nikolaev VO, El-Hamamsy I, Potter CM, Wright P, Kadir SH, Lyon AR, Mitchell JA, Chester AH, Klenerman D, Lab MJ, Korchev YE, Harding SE, Gorelik J - J R Soc Interface (2011)

Bottom Line: At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio.The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents.In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Science, National Heart and Lung Institute, Imperial College London, , Dovehouse Street, London SW36LY, UK.

ABSTRACT
Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

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Aortic valve architecture. (a) Surface topography of a live explanted porcine aortic valve demonstrating cell shape, size and alignment using scanning ion conductance microscopy (SICM) (A. Moshkov 2010, unpublished data). Effective pixel width 313 nm, scan duration 23 min. Scanning pipette had resistance of 100 MΩ and estimated tip diameter of 100 nm. (b) Glutaraldehyde-fixed sample of valve imaged using scanning electron microscopy, 2000×, showing cell shape and alignment similar to SICM image in (a). Scale bar, 5 µm. (Scanning electron microscope image courtesy of Dr Adrian H. Chester, Cardiovascular Science, Harefield Hospital, Imperial College London, London, UK.)
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RSIF20100597F2: Aortic valve architecture. (a) Surface topography of a live explanted porcine aortic valve demonstrating cell shape, size and alignment using scanning ion conductance microscopy (SICM) (A. Moshkov 2010, unpublished data). Effective pixel width 313 nm, scan duration 23 min. Scanning pipette had resistance of 100 MΩ and estimated tip diameter of 100 nm. (b) Glutaraldehyde-fixed sample of valve imaged using scanning electron microscopy, 2000×, showing cell shape and alignment similar to SICM image in (a). Scale bar, 5 µm. (Scanning electron microscope image courtesy of Dr Adrian H. Chester, Cardiovascular Science, Harefield Hospital, Imperial College London, London, UK.)

Mentions: The aortic valve is composed of a monolayer of endothelial cells lining both sides of the valve, with a mixed population of interstitial cells (smooth muscle cells, fibroblasts and myofibroblasts) lying in between. This is all in a complex haemodynamic and mechanical environment, with endothelial cells from both sides of the valve exposed to different shear stresses [30]. Detailed in situ investigations in this topic would be extremely valuable, to understand the pathophysiology and eventual therapies. Figure 2 shows that the SICM can uniquely provide in situ evaluation of the topography of aortic valve endothelial cells from the ventricular side of the valve on freshly explanted unfixed aortic valve specimens. The resolution of our SICM-acquired valve topography images was close to that of electron microscopy analysis of the same tissue, which was previously fixed and shaded (figure 2b).Figure 2.


Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells.

Miragoli M, Moshkov A, Novak P, Shevchuk A, Nikolaev VO, El-Hamamsy I, Potter CM, Wright P, Kadir SH, Lyon AR, Mitchell JA, Chester AH, Klenerman D, Lab MJ, Korchev YE, Harding SE, Gorelik J - J R Soc Interface (2011)

Aortic valve architecture. (a) Surface topography of a live explanted porcine aortic valve demonstrating cell shape, size and alignment using scanning ion conductance microscopy (SICM) (A. Moshkov 2010, unpublished data). Effective pixel width 313 nm, scan duration 23 min. Scanning pipette had resistance of 100 MΩ and estimated tip diameter of 100 nm. (b) Glutaraldehyde-fixed sample of valve imaged using scanning electron microscopy, 2000×, showing cell shape and alignment similar to SICM image in (a). Scale bar, 5 µm. (Scanning electron microscope image courtesy of Dr Adrian H. Chester, Cardiovascular Science, Harefield Hospital, Imperial College London, London, UK.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104336&req=5

RSIF20100597F2: Aortic valve architecture. (a) Surface topography of a live explanted porcine aortic valve demonstrating cell shape, size and alignment using scanning ion conductance microscopy (SICM) (A. Moshkov 2010, unpublished data). Effective pixel width 313 nm, scan duration 23 min. Scanning pipette had resistance of 100 MΩ and estimated tip diameter of 100 nm. (b) Glutaraldehyde-fixed sample of valve imaged using scanning electron microscopy, 2000×, showing cell shape and alignment similar to SICM image in (a). Scale bar, 5 µm. (Scanning electron microscope image courtesy of Dr Adrian H. Chester, Cardiovascular Science, Harefield Hospital, Imperial College London, London, UK.)
Mentions: The aortic valve is composed of a monolayer of endothelial cells lining both sides of the valve, with a mixed population of interstitial cells (smooth muscle cells, fibroblasts and myofibroblasts) lying in between. This is all in a complex haemodynamic and mechanical environment, with endothelial cells from both sides of the valve exposed to different shear stresses [30]. Detailed in situ investigations in this topic would be extremely valuable, to understand the pathophysiology and eventual therapies. Figure 2 shows that the SICM can uniquely provide in situ evaluation of the topography of aortic valve endothelial cells from the ventricular side of the valve on freshly explanted unfixed aortic valve specimens. The resolution of our SICM-acquired valve topography images was close to that of electron microscopy analysis of the same tissue, which was previously fixed and shaded (figure 2b).Figure 2.

Bottom Line: At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio.The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents.In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Science, National Heart and Lung Institute, Imperial College London, , Dovehouse Street, London SW36LY, UK.

ABSTRACT
Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

Show MeSH
Related in: MedlinePlus