Figure 6.

Multiple fluorophores can be used within an experiment, but the pinhole size should be kept at the same absolute diameter. A. A cell was sequentially injected with Alexa Fluor 555 (dendrite in red, top panel) and Alexa Fluor 488 (dendrite in green, middle panel). The bottom panel shows the dendrite when the two labels are superimposed, with yellow representing voxels containing both dyes. B. Images of the same dendritic segments were taken with the same relative pinhole (diameter set to 1 AU for each respective dye) or with the same absolute pinhole size (setting the pinhole diameter to 1 AU for Alexa Fluor 488 and maintaining it at the same diameter for Alexa Fluor 555, which resulted in a 0.88 AU pinhole for the red dye). Individual spine head volumes were measured using NeuronStudio following appropriate deconvolution of each fluorophore. Spine head volumes differed significantly when the same relative pinhole diameter was used (paired t-test, p<1×10⁻⁵), but not when the physical diameter of the pinhole was kept constant (paired t-test, p=0.6). Each colored line represents the volume of an individual spine head, while the black line represents the average volume of all measured spines. Animal use in this experiment was conducted with strict adherence to our Institutional Animal Care and Use Committee.