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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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(a) Illustration of the use of SICM for electrophysiological measurement. (i) A scan of a region of neonatal rat ventricular myocytes monolayer with highest thickness = 12 µm. (ii) Representative resting Vm measured with SICM (n = 20). (iii) Scan of a monolayer of cardiac myofibroblasts. Note that the highest thickness (6 µm) corresponds to the region above the nuclei. (iv) SICM allowed successful measurement of Vm in the region of the cell surface above the nucleus (n = 20). Pipette had a resistance of approximately 20 MΩ and an estimated tip diameter of approximately 500 nm. Effective pixel width in topography images was 400 nm, scan duration 20 min. (b) Whole-cell recording in neonatal rat ventricular myocytes using SICM. (i) Resistance of the pipette used for whole-cell recording. The distribution of the seal resistance RSEAL measured after obtaining stable gigaseal configuration (solid squares). (ii) Schematic of a patch-pipette performing whole-cell recording in a neonatal rat ventricular myocyte. (iii) Example of a whole-cell action potential recording in a neonatal rat ventricular myocytes in the current-clamp mode showing spontaneous action potential firing. Values were corrected for liquid junction potential (n = 42). Pipettes used for whole-cell patch-clamp recording had resistance in the range of 6–9 MΩ and estimated diameter of 1.7–1.1 µm diameter. (M. Miragoli 2009 & P. Novak 2010, unpublished data.) (Online version in colour.)
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RSIF20100597F7: (a) Illustration of the use of SICM for electrophysiological measurement. (i) A scan of a region of neonatal rat ventricular myocytes monolayer with highest thickness = 12 µm. (ii) Representative resting Vm measured with SICM (n = 20). (iii) Scan of a monolayer of cardiac myofibroblasts. Note that the highest thickness (6 µm) corresponds to the region above the nuclei. (iv) SICM allowed successful measurement of Vm in the region of the cell surface above the nucleus (n = 20). Pipette had a resistance of approximately 20 MΩ and an estimated tip diameter of approximately 500 nm. Effective pixel width in topography images was 400 nm, scan duration 20 min. (b) Whole-cell recording in neonatal rat ventricular myocytes using SICM. (i) Resistance of the pipette used for whole-cell recording. The distribution of the seal resistance RSEAL measured after obtaining stable gigaseal configuration (solid squares). (ii) Schematic of a patch-pipette performing whole-cell recording in a neonatal rat ventricular myocyte. (iii) Example of a whole-cell action potential recording in a neonatal rat ventricular myocytes in the current-clamp mode showing spontaneous action potential firing. Values were corrected for liquid junction potential (n = 42). Pipettes used for whole-cell patch-clamp recording had resistance in the range of 6–9 MΩ and estimated diameter of 1.7–1.1 µm diameter. (M. Miragoli 2009 & P. Novak 2010, unpublished data.) (Online version in colour.)

Mentions: The use of a glass pipette containing an electrode connected to an amplifier immediately calls for the application of other commonly used electrophysiological techniques such as patch-clamp and intracellular voltage measurements. The SICM is perfectly suited for both techniques and it further improves their performance. Two main reasons place SICM as an ideal instrument for intracellular measurement: (i) precise determination of the cell morphology before impalement and (ii) nanometric, automatic and vertical approach. The SICM permits the selection of the location on the cell surface by a well-controlled vertical approach with nanometre precision, resulting in the easy formation of a contact gigaseal with the membrane bilayer. Figure 7a(i) (scan time 7 min) shows a 50 × 50 µm topographical scan of neonatal rat ventricular myocytes with a tallest peak of 12 µm. Using a pipette filled with 3 mol l−1 KCl and the precise three-dimensional position control of the SICM, we could place the pipette at any place on the cell surface. When lowering the pipette, the access resistance started to increase, indicating a ‘quasi-attachment’ onto the cell surface. A small additional, automatic downward advancement of 50–100 nm resulted in the pipette tip penetrating the membrane. After 1 min of stabilized impalement, Vm was recorded (figure 7a). In this example, resting Vm, as expected, was −79 mV and the cardiac monolayer showed spontaneous electrical activity with depolarizing transients.Figure 7.


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)

(a) Illustration of the use of SICM for electrophysiological measurement. (i) A scan of a region of neonatal rat ventricular myocytes monolayer with highest thickness = 12 µm. (ii) Representative resting Vm measured with SICM (n = 20). (iii) Scan of a monolayer of cardiac myofibroblasts. Note that the highest thickness (6 µm) corresponds to the region above the nuclei. (iv) SICM allowed successful measurement of Vm in the region of the cell surface above the nucleus (n = 20). Pipette had a resistance of approximately 20 MΩ and an estimated tip diameter of approximately 500 nm. Effective pixel width in topography images was 400 nm, scan duration 20 min. (b) Whole-cell recording in neonatal rat ventricular myocytes using SICM. (i) Resistance of the pipette used for whole-cell recording. The distribution of the seal resistance RSEAL measured after obtaining stable gigaseal configuration (solid squares). (ii) Schematic of a patch-pipette performing whole-cell recording in a neonatal rat ventricular myocyte. (iii) Example of a whole-cell action potential recording in a neonatal rat ventricular myocytes in the current-clamp mode showing spontaneous action potential firing. Values were corrected for liquid junction potential (n = 42). Pipettes used for whole-cell patch-clamp recording had resistance in the range of 6–9 MΩ and estimated diameter of 1.7–1.1 µm diameter. (M. Miragoli 2009 & P. Novak 2010, unpublished data.) (Online version in colour.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSIF20100597F7: (a) Illustration of the use of SICM for electrophysiological measurement. (i) A scan of a region of neonatal rat ventricular myocytes monolayer with highest thickness = 12 µm. (ii) Representative resting Vm measured with SICM (n = 20). (iii) Scan of a monolayer of cardiac myofibroblasts. Note that the highest thickness (6 µm) corresponds to the region above the nuclei. (iv) SICM allowed successful measurement of Vm in the region of the cell surface above the nucleus (n = 20). Pipette had a resistance of approximately 20 MΩ and an estimated tip diameter of approximately 500 nm. Effective pixel width in topography images was 400 nm, scan duration 20 min. (b) Whole-cell recording in neonatal rat ventricular myocytes using SICM. (i) Resistance of the pipette used for whole-cell recording. The distribution of the seal resistance RSEAL measured after obtaining stable gigaseal configuration (solid squares). (ii) Schematic of a patch-pipette performing whole-cell recording in a neonatal rat ventricular myocyte. (iii) Example of a whole-cell action potential recording in a neonatal rat ventricular myocytes in the current-clamp mode showing spontaneous action potential firing. Values were corrected for liquid junction potential (n = 42). Pipettes used for whole-cell patch-clamp recording had resistance in the range of 6–9 MΩ and estimated diameter of 1.7–1.1 µm diameter. (M. Miragoli 2009 & P. Novak 2010, unpublished data.) (Online version in colour.)
Mentions: The use of a glass pipette containing an electrode connected to an amplifier immediately calls for the application of other commonly used electrophysiological techniques such as patch-clamp and intracellular voltage measurements. The SICM is perfectly suited for both techniques and it further improves their performance. Two main reasons place SICM as an ideal instrument for intracellular measurement: (i) precise determination of the cell morphology before impalement and (ii) nanometric, automatic and vertical approach. The SICM permits the selection of the location on the cell surface by a well-controlled vertical approach with nanometre precision, resulting in the easy formation of a contact gigaseal with the membrane bilayer. Figure 7a(i) (scan time 7 min) shows a 50 × 50 µm topographical scan of neonatal rat ventricular myocytes with a tallest peak of 12 µm. Using a pipette filled with 3 mol l−1 KCl and the precise three-dimensional position control of the SICM, we could place the pipette at any place on the cell surface. When lowering the pipette, the access resistance started to increase, indicating a ‘quasi-attachment’ onto the cell surface. A small additional, automatic downward advancement of 50–100 nm resulted in the pipette tip penetrating the membrane. After 1 min of stabilized impalement, Vm was recorded (figure 7a). In this example, resting Vm, as expected, was −79 mV and the cardiac monolayer showed spontaneous electrical activity with depolarizing transients.Figure 7.

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