Introduction 19 15 4 17 1 6 17 25 6 20 26 25 5 2 On the other hand, electron microscopy techniques have more than sufficient resolution and provide much more detailed information about the surface topography than optical images since the cell membrane can be imaged. However, these require fixation and specialised treatments of the cells, which potentially can alter the structure of interest and are thus incompatible with live studies. 5 9 13 14 7 23 Materials and methods SSCM 14 23 z 1 z x y 22 Fig. 1 a b c d e top bottom f top bottom  The SCIM scanning head was developed in collaboration with Ionscope Limited, UK and mounted on a Nikon TE2000-U Inverted Microscope (Nikon Co., Japan). The sample holder was attached to a 100-μm HERA XY Nanopositioning System (Physik Instrumente (PI) GmbH & Co., Germany) used for lateral scanning. Vertical measurement and modulation was provided by 12-μm LISA XY Nanopositioning System (Physik Instrumente (PI) GmbH & Co., Germany). Both piezo stages were mounted on 25-mm translation stage DC motors (Physik Instrumente (PI) GmbH & Co., Germany) to provide coarse lateral and vertical approach. The setups were controlled via a computer with a SBC6711 DSP board equipped with A4D4 ADC/DAC modules (Innovative Integration, USA) using SICM software v. 1.2.00 (Ionscope Limited). The time to acquire a 512 × 512 pixel image was approximately 10 min. Two types of nanopipettes were used for the experiments. For low resolution images, nanopipettes with internal diameters ~150 nm were pulled from borosilicate glass capillaries. High resolution imaging was made using quartz nanopipettes with internal diameters ~70 nm. The nanopipettes were made from 1.00-mm outer diameter by 0.5-mm inner diameter capillaries with inner filament (Sutter Instrument, USA) using a laser-based Brown–Flaming puller (model P-2000, Sutter Instrument, San Rafael, CA, USA). The nanopipettes, backfilled with phosphate-buffered saline (PBS) and lowered in PBS, produced a resistance of approximately 300 MΩ for quartz and 100 MΩ for borosilicate pipettes. The maximum ion current measured using an Axopatch 200B (Axon Instruments, USA) was ~0.7 nA for quartz and ~1.5 nA for borosilicate pipettes. The set-point for imaging was 1% of the maximum of modulated ion current. The excitation light source was provided by a GPNT-02 laser diode (532-nm wavelength, IQ1A 635-nm laser; Power Technology Inc., USA). The optical recording system consisted of a Nikon TE2000-U Inverted Microscope equipped with a ×100 1.3-N.A. oil-immersion objective. The excitation light was fed through an epi-fluorescent filter block and emitted light was collected by a photomultiplier with a pinhole (model D-104-814; Photon Technology International, UK). Image processing and data analysis Matching VLP topographical structure to its corresponding fluorescent signal was done as follows: fluorescent confocal images were threshold to subtract the background, and the positions of individual fluorescent spots were marked by arrowheads and the multiple spots (where individual signals could not be resolved) circled. All positional markers were then grouped into one template and placed over the simultaneously recorded topographical image. As the result of this procedure, those topographical features having corresponding fluorescent signals were marked. 10 18 21 Cell culture and plasmids 2 Transfection procedure 6 4 Results Imaging of the cell surface using SICM 1 Materials and methods 9 13 14 8 1 1 1 1 1 1 1 Fig. 2 a b c b d b c e f e g f g h  Scanning surface confocal microscopy In order to identify what pit indentations detected topographically by SICM corresponded to what endocytic pits, clathrin or caveolin, we transfected Cos-7 cells with corresponding GFP construct and then used scanning surface confocal microscopy to study the transfected cells. 2 7 Imaging clathrin-coated pits in membranes of fixed cells using SSCM 2 2 2 2 2 2 16 5 Imaging caveolae in membranes of fixed cells using SSCM 3 3 3 Fig. 3 a b a c a b d e d f g d dotted square h g i h e dotted square  3 3 3 15 25 25 Imaging flotillin in membranes of fixed cells using SSCM 1 6 4 4 4 4 4 4 4 4 Fig. 4 a b a c a b d e d f e white arrows g solid arrows hollow arrows h g i g arrows Hollow arrows  Imaging endocytic pits in membranes of living cells using SSCM 5 Fig. 5 a b a c a d  5 Discussion By combining high resolution ion conductance imaging of the cell surface topography with fluorescence confocal imaging, we can identify the molecular nature of endocytic pits on the surface of living cells and measure the topography of the pits. For the first time, we showed that flotillin 1 and 2 is involved in the formation of ~200-nm-size indentations in the cell membrane. This observation is important evidence in support of the involvement of this protein in clathrin- and caveolin-independent endocytosis. 11 24 3 12 We have also shown that it is possible to apply our method to live cells. The clathrin-coated pits show fast dynamics on the time scale of our current imaging but suggest that improvements in the speed of imaging should allow us to follow the dynamics of endocytic pits on living cells with the intriguing possibility of directly imaging the endocytic process in real time.