title: Angular Approach Scanning Ion Conductance Microscopy.
abstract: Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology.
authors.affiliation: Department of Medicine, Imperial College London, London, United Kingdom. Electronic address: a.shevchuk@ic.ac.uk.
authors.forename: Andrew
authors.initials: A
authors.lastname: Shevchuk
authors.affiliation: Rayne Institute, King's College London, London, United Kingdom.
authors.forename: Sergiy
authors.initials: S
authors.lastname: Tokar
authors.affiliation: Department of Medicine, Imperial College London, London, United Kingdom; Department of Materials and Department of Bioengineering and Institute for Biomedical Engineering, Imperial College London, London, United Kingdom.
authors.forename: Sahana
authors.initials: S
authors.lastname: Gopal
authors.affiliation: National Heart and Lung Institute and Department of Cardiac Medicine, Imperial Center for Translational and Experimental Medicine, Imperial College London, London, United Kingdom.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Sanchez-Alonso
authors.affiliation: Oxford Centre for Diabetes, Endocrinology, and Metabolism, University of Oxford, Churchill Hospital, Oxford, United Kingdom.
authors.forename: Andrei I
authors.initials: AI
authors.lastname: Tarasov
authors.affiliation: Department of Physiology, University of Kentucky, Lexington, Kentucky.
authors.forename: A Catalina
authors.initials: AC
authors.lastname: Vélez-Ortega
authors.affiliation: Department of Materials and Department of Bioengineering and Institute for Biomedical Engineering, Imperial College London, London, United Kingdom.
authors.forename: Ciro
authors.initials: C
authors.lastname: Chiappini
authors.affiliation: Oxford Centre for Diabetes, Endocrinology, and Metabolism, University of Oxford, Churchill Hospital, Oxford, United Kingdom.
authors.forename: Patrik
authors.initials: P
authors.lastname: Rorsman
authors.affiliation: Department of Materials and Department of Bioengineering and Institute for Biomedical Engineering, Imperial College London, London, United Kingdom.
authors.forename: Molly M
authors.initials: MM
authors.lastname: Stevens
authors.affiliation: National Heart and Lung Institute and Department of Cardiac Medicine, Imperial Center for Translational and Experimental Medicine, Imperial College London, London, United Kingdom.
authors.forename: Julia
authors.initials: J
authors.lastname: Gorelik
authors.affiliation: Department of Physiology, University of Kentucky, Lexington, Kentucky.
authors.forename: Gregory I
authors.initials: GI
authors.lastname: Frolenkov
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
authors.affiliation: Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Yuri E
authors.initials: YE
authors.lastname: Korchev
mesh_terms: D000328:Adult; D000818:Animals; D002478:Cells, Cultured; D003470:Culture Media; D004867:Equipment Design; D005260:Female; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods*; D008297:Male; D051379:Mice; D008846:Micromanipulation / Q000295:instrumentation / Q000379:methods; D008855:Microscopy, Electron, Scanning; D020527:Microscopy, Scanning Probe / Q000295:instrumentation / Q000379:methods*; D036103:Nanotechnology; D018408:Patch-Clamp Techniques / Q000295:instrumentation / Q000379:methods; D017207:Rats, Sprague-Dawley
chemical_list: D003470:Culture Media