The ribbon synapse of cochlear inner hair cells (IHCs) is the first relay point of the auditory system. Each contact made by a single IHC onto the afferent nerve can operate with a high degree of temporal precision. To understand the underlying mechanisms, we have developed techniques to image vesicle release at the IHC ribbon using the styryl dye FM1-43 in a manner similar to that reported previously (Griesinger et al., 2005), but now adapted to the mouse in order to permit access to a wide range of transgenic models with altered synaptic function. By voltage-clamping the IHC we show that it is also possible to control the calcium currents triggering vesicle release. Mouse cochleas were imaged in situ in the isolated temporal bone after their removal in accordance with current national guidelines. The bone was attached to the base of a chamber containing artificial perilymph (in mM): NaCl,140; KCl,4; CaCl₂,2; MgCl₂,1.5; Hepes, 10 to pH 7.3). A small opening made in the apical turn exposed the 10-20kHz region of the organ of Corti. All ages of preparation can be used. FM1-43 (2 μM) was added to the bath to be taken up rapidly by IHCs from their intact apical surface and trafficked to the cell base at an effective rate of 0.08 μm-s^-1. After 300 s regions corresponding to ribbon sites were identified using a 2 photon (2P, 840nm) laser scanning microscope. Images of FM1-43 ‘hotspots’ were acquired as a series of frames (61 ms/frame) or as lines scans (1.8 ms/line). On stimulation by transepithelial extracellular current designed to depolarise the basolateral terminal, hotspots destained by up to 15% during 200ms 160 μA pulses. This result is consistent with vesicle exocytosis. IHCs were also stimulated during conventional whole-cell tight-seal recording from the basolateral surface. The pipette contained 140 mM Cs⁺ to reduce large outward K⁺ currents. Under such conditions inward calcium currents of up to 80 pA, with peak currents at -15mV, were recorded. Simultaneous 2P imaging showed FM1-43 localised at the presumed ribbon sites. Cell depolarization by 60mV (from V_h = – 70 mV) decreased the fluorescence by 1-2%. The fluorescence intensity recovered with a time constant of 220 ms. In a few cases there was also a small fluorescence rebound, suggesting redistribution of dye. The imaged destaining rate is consistent with the inference (Goutman & Glowatzki, 2007) that only a small number of vesicles (10-20) may be released at each site on short 200 ms depolarizing commands.