Figure 1.

Pixel and optical resolution for calcium imaging in the adult frog neuromuscular junction. A, Diagram of the adult frog neuromuscular junction. B, Magnified active zones (boxed region in A) with highly organized active zone proteins, which include voltage-gated Ca²⁺ channels, and docked synaptic vesicles. The estimated relationship between active zone dimensions, pixel size, and optical resolution are also shown (left). For comparison, a composite image that includes fluorescent staining of postsynaptic acetylcholine receptors (background, grayscale) overlaid with Ca²⁺-evoked fluorescent signals after nerve stimulation (foreground, false color) is shown on the right. Scale bar, 1 μm. C, Experimentally measured point spread function of the optical system from a fluorescent bead with diameter of 0.02 μm. The Gaussian fit (red) has a width at half-amplitude of 0.45 μm. We thus estimated that approximately half the length of a single active zone can be resolved optically. D, Correlation coefficient of neighboring pixels that detected AP-evoked fluorescence signals after repeated low-frequency nerve stimulus tightly depends on the distance between pixels. The Lorentzian fit (red) has a width at half-amplitude of 0.41 μm (points represent mean ± SD; n = 4361 pixels). These data are fit best with a Lorentzian function, rather than a Gaussian, likely because of the presence of some signal contributing from neighboring sites, especially at the tails of the distribution. E, Predicted active zone regions of a representative frog motor nerve terminal identified by labeling the postsynaptic acetylcholine receptor clusters with Alexa Fluor 594–α-bungarotoxin. F, Representative difference images showed a large trial-to-trial variability in the spatial distribution of Ca²⁺ entry evoked by single APs. Scale bar, 2 μm (applies to E, F).