In classical microscopy the resolution is limited to ~200-300 nm due to Abbe’s diffraction limit. In the past two decades several techniques emerged to overcome this limit, among which the STimulated Emission Depletion (STED) microscopy has attracted wide interest (1). In STED microscopy, two beams are required: one excites the sample as in classical confocal microscopy, while the other one, with a donut-shape profile, de-excites the fluorophores by stimulated emission, which avoids the fluorescence emission. Only the tiny volume located at the center of the donut will emit fluorescence. The size of this emitting spot defines the resolution of the STED images, and strongly depends on the quality and the intensity of the STED beam that is critical to have zero energy at the center of the donut. We built a resonant scanning STED microscope, which achieves a lateral resolution in biological samples of ~30 nm in a large field of view of ~40 x 40 µm acquired at ~10 nm/pixel. To acquire images of ~4000 x 4000 points at a speed of 16,000 lines per second, we used an 8 KHz resonant scanning mirror synchronized with a 96 MHz (10.4 ns/point) AD converter. Light was collected by a fast and ultrasensitive photomultiplier (H7422-PA-40, Hamamatsu Photonics) (for more details see: http://www.anes.ucla.edu/sted/index.html). Potassium and calcium channels in cardiomyocytes and in isolated cardiac mitochondria were immunolabeled with specific antibodies and Atto-647N as the secondary antibody. Sprague-Dawley rats or C57BL/6 were used for organelle/cell isolation and protocols received institutional approval. At fast scanning speed, the photobleaching of samples was considerably reduced allowing repeated imaging of the same area, as well as the acquisition of a stack of planes for 3D reconstruction. With this microscope, we have uncovered a discrete distribution of BK channels within a single mitochondrion, and a distinct array of L-type calcium channels and ryanodine receptors in cardiomyocytes in control and after heart failure. For mitochondrial BK channels, a cluster size histogram revealed a peak at ~50 nm (500 individual clusters from three independent mitochondrial preparations). In conclusion, we have constructed a STED microscope that allows the detailed analysis of protein clusters using a large field of view.