We describe a major advance in scanning ion conductance microscopy: a new hopping mode that allows non-contact imaging of the complex surfaces of live cells with resolution better than 20 nm. The effectiveness of this novel technique was demonstrated by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allows studying nanoscale phenomena on the surface of live cells under physiological conditions.There is a great interest in developing methods to image live cells at nanoscale resolution. Scanning probe microscopy (SPM) is one approach to this problem and both atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) have been used to image live cells 1,2 . However, deformation of the soft and responsive cell by AFM cantilever, particularly when imaging eukaryotic cells, represents a substantial problem for AFM. SECM, in contrast, involves no physical contact with the sample but a true topographic imaging of the convoluted surface of living cells with nanoscale resolution has not been yet achieved. Scanning ion conductance microscopy (SICM) 3 is another form of SPM, that allows imaging of the cell surface under physiological conditions without physical contact and with a resolution of 3-6 nm 4,5 . However, until now, SICM was restricted to imaging relatively flat surfaces, like all other SPM techniques. This is because when the probe encounters a vertical structure, it inevitably collides with the specimen (Fig. 1a). Here we report a novel mode of SICM that allows imaging of even the most convoluted surface structures at the nanoscale. SICM is based on the phenomenon that the ion flow through a sharp fluid-filled nanopipette is partially occluded when the pipette approaches the surface of a cell 3 . In conventional SICM a nanopipette is mounted on a three-dimensional piezo and automatic feedback control moves the pipette up or down to keep the pipette current at a constant level (the set point), while the sample is scanned in X-Y directions. Thus, a pipette-sample separation, typically equal to the pipette's inner radius, is maintained during imaging. In our hopping probe ion conductance microscopy (HPICM), we no longer use continuous feedback. Instead, at each imaging point, the pipette approaches the sample from a starting position that is above any of the surface features (Fig. 1b). The reference current is measured while the pipette is well away from the surface. The pipette then approaches until the current is reduced by a predefined amount, usually 0.25-1%. The Z-position when the current achieves this reduction is recorded as the height of the sample at this imaging point. Typically, even at a 1% reduction of the current, the pipette is still at a distance of about one inner pipette radius from the surface. Therefore, the probe never touches the surface of the cell. The pipette is then withdrawn away from the surface and the sample moved laterally to determine the next imaging point. By continuously upd...

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T-tubular invaginations of the sarcolemma of ventricular cardiomyocytes contain junctional structures functionally coupling Ltype calcium channels to the sarcoplasmic reticulum calciumrelease channels (the ryanodine receptors), and therefore their configuration controls the gain of calcium-induced calcium release (CICR). Studies primarily in rodent myocardium have shown the importance of T-tubular structures for calcium transient kinetics and have linked T-tubule disruption to delayed CICR. However, there is disagreement as to the nature of T-tubule changes in human heart failure. We studied isolated ventricular myocytes from patients with ischemic heart disease, idiopathic dilated cardiomyopathy, and hypertrophic obstructive cardiomyopathy and determined T-tubule structure with either the fluorescent membrane dye di-8-ANNEPs or the scanning ion conductance microscope (SICM). The SICM uses a scanning pipette to produce a topographic representation of the surface of the live cell by a non-optical method. We have also compared ventricular myocytes from a rat model of chronic heart failure after myocardial infarction. T-tubule loss, shown by both ANNEPs staining and SICM imaging, was pronounced in human myocytes from all etiologies of disease. SICM imaging showed additional changes in surface structure, with flattening and loss of Z-groove definition common to all etiologies. Rat myocytes from the chronic heart failure model also showed both T-tubule and Z-groove loss, as well as increased spark frequency and greater spark amplitude. This study confirms the loss of T-tubules as part of the phenotypic change in the failing human myocyte, but it also shows that this is part of a wider spectrum of alterations in surface morphology.calcium handling ͉ heart failure ͉ morphology ͉ T-tubule