Scanning Ion Conductance Microscopy

SICM History

The Scanning Ion Conductance Microscope (SICM) was originally developed and described by Prof. P.K. Hansma and co-authors at the University of California (Hansma et al., 1989). SICM belongs to a wide family of scanning probe microscopes (SPM), and it was specially designed for the scanning of soft non-conductive materials at sub-micrometer resolution that are bathed in electrolyte solution. Also, in order to achieve higher contrast imaging, SICM was used in combination with tapping mode atomic force microscopy (Proksch et al., 1996). However, for a long time this technique was limited to imaging of flat polymeric films. In 1997 significant improvements to SICM have been made by Y. E. Korchev et. al to allow the imaging of live cells without making direct contact with the sample surface (Korchev et al., 1997a).

 

SICM Principles of Operation

   

The SICM consists of a glass micropipette probe filled with electrolyte lowered into a bath of electrolyte (see diagram) toward an insulating, for ions, surface of the sample. As the tip of the micropipette approaches the sample, the ion conductance reduces because the gap that ions can flow through is decreased. Changes of the ion current are measured by an ion current amplifier, and are used as a feedback input signal by scanner control unit to keep the distance between pipette tip and sample constant by applying corresponding voltages to the Z-piezo drive during the scanning procedure. Therefore, the path of the tip follows the topography of the surface.

 

SICM Applications

 

We have recently pioneered the development of an array of new and powerful biophysical tools that allow quantitative measurements and non-invasive functional imaging of single protein molecules in living cells. Scanning ion conductance microscopy and a battery of associated innovative methods are unique among current imaging techniques (see left figure), not only in spatial resolution of living and functioning cells, but also in the rich combination of imaging with other functional and dynamical interrogation methods. These methods, crucially, will facilitate the study of integrated nano-behaviour in living cells in health and disease.

The family of techniques we have developed is shown in the figures below.

                 

The methodologies are applicable to a wide variety of different cell types and tissues and we have applied these methods to neuro, cardiac and sperm physiology, endocrinology and virology. Our key achievements are:

 

1. The development of a unique non-contact system for imaging living cell surfaces and their dynamics down to the level of individual protein complexes. The method is based on a scanned nanopipette, using ion conductance feedback to maintain constant pipette-sample distance. We can interrogate unfixed, unstained, living cells with high resolution. This enabled us to:

• · Imaging protein complexes on a live cell.

• · Determine the dynamics of microvilli and how they can be used as building blocks to assemble more complex structures.
• · High resolution Hopping mode imaging.

• · Develop an in-vitro model of rhythm disturbance in heart and study bile acid effects on cardiac myocyte function.

Refs:       Kidney Int. (2005) 68:1071-1077; Int J Obstet Gynaecol (2004) 111:867-70; P.N.A.S. (2003) 100: 5819-5822; FEBS Lett. (2003) 548: 74-78; Int J Obstet Gynaecol (2003) 110: 467-74; Clin Sci (Lond). (2002) 103: 191-200; Biophys. J. (2000) 78: 451-457; Angew. Chem. Int. Ed Engl. 45:2212-2216; Nature Meth. (2009; FASEB J. (2009); Nature Meth. (2010), 7, 170

1. Functional imaging and “Smart” patch clamp where we can record single ion channel activity with molecular size precision at defined positions on the cell surface including hitherto inscrutable regions such as the synapse of dendritic neurons, specific regions of a sperm, and the t-tubule of cardiac myocytes. We have thus:

• · Functionally mapped the distribution of ion channels on cardiac myocytes revealing possible action potential structure-function mechanisms.

• · Identified a plausable mechanism of action of aldosterone on kidney cells.

Refs:       Dev Biol (2004) 274: 308-17; Meth Cell Biol. (2004) 74:545-576; Mol. Cel. Endocrinol. (2004) 217: 101-108; Biophys. J. (2002) 83, 3296–3303; Faseb J. (2002) 16: 748-751; Nature Cell Biol. (2000) 2: 616-619; Proc. Natl. Acad. Sci. U. S. A (2005) 102:15000-15005; Am. J. Physiol Renal Physiol (2007) 292:F1734-F1740; Biophys. J. (2008) 94:1646-1655; Science (2010)

1. The combination of high resolution topographic imaging with two colour fluorescence for simultaneous mapping topography and specific fluorophore labelled proteins. This has been used to:

• · Follow viral entry in real-time, and identify viral entry pathway into living cells.

• Follow endocytic pathways.
• Monitor membrane and Ca²⁺ dynamics.

Refs:       P.N.A.S. (2002) 99, 16018–16023; Biophys. J. (2001) 81: 1759-1764; J Microsc. (2003) 209: 94-101; Anal Chem. (2002) 74: 2612-2616; Clinical Sci. (2001) 100: 363-369; Biophys. J. 94:4089-4094; Pflugers Arch. 456:227-235

1. Control the delivery of small molecules, proteins and antibodies to defined positions on the cell surface. This can be used to for minimal staining of protein complexes to study surface diffusion of molecules, or any other cell manipulation, with molecular size precision. In consequence, we have:

• · Controlled the deposition of biological molecules using a nanopipette for a combination of top-down and bottom-up assembly of novel structures and the formation of nanoarrays of biological molecules.·

          

Ref:        J Am Chem Soc. (2004) 126: 6508-9; Biophys J. (2004) 86:1018-1027; J. Am. Chem. Soc. (2003) 125: 9834 – 9839; J Am Chem Soc. (2002) 124: 8810-8811; Anal Chem (2002) 74: 1380-1385; Angew. Chem. Int. Ed Engl. 44:6854-6859;

 

• Development of novel non-contact mechanical measurement technique.

 

Ref:        Biophys. J. (2008) 95(6): 3017-3027