Figure 5.

The bleaching rate as a function of laser power varies dramatically among fluorophores and should therefore be determined empirically prior to start of imaging. Five consecutive 2D images were taken with 50 nm voxels, frame average of 4, speed of 2.5 μs/pixel, and 1 AU relative pinhole diameter. The peak intensity of 3–5 individual spines from 2–3 time series per fluorophore was measured in the first and last frame in order to calculate the percentage bleaching. Note that slower bleaching rate is not necessarily an indication of a brighter fluorophore (where brightness is defined as photons emitted per molecule per unit time), and should therefore not be used in as a factor in the choice of dye for microinjection. Rather, the bleaching rate should be determined in order to establish the optimal power for the chosen fluorophore and imaging parameters. Use a lower power (allow no more than 3–5% bleaching using the metric described here) if significant oversampling in the Z-dimension will be used, and a higher power (allow up to 10% bleaching) if imaging at or near a Nyquist Z-step optimal size. Animal use in this experiment was conducted with strict adherence to our Institutional Animal Care and Use Committee.