Cortical neurons transduced with relevant Lentivirus as described above were imaged at DIV 15 (7 days after iCre transfection) in imaging solution (HBSS with 2 mM GlutaMAX, 1 mM sodium pyruvate, and 10 mM HEPES pH 7.4). As a membrane-localized reference channel, Crimson RFP26 (link) with a C-terminal farnesylation motif (CAAX) for membrane targeting was co-expressed with an ASAP variant via IRES. The epifluorescence from the cells were imaged on an Axiovert 200M inverted microscope with a 40x 1.2-NA water-immersion objective (Zeiss) and an X-Cite 120 metal-halide lamp (Exfo) as the excitation light source. For ASAP, excitation and emission filters were BP450-490 and BP515-565 (Zeiss). For Crimson, excitation and emission filters were HQ535/50m and HQ625/60m (Chroma). For each cell, 15 focal planes spaced 1 μm apart were captured by a Flash4.0LT+ camera using μManager. To quantify membrane localization, a custom-written MATLAB (MathWorks) code was used. Membrane masks of the neurons were generated by applying the graythresh function on Sobel-filtered red channel images. The soma masks were manually selected using the roipoly function on red channel images. Then the two masks were applied to background-subtracted green channel images to obtain the membrane fluorescence and the soma fluorescence of the ASAP reporter. The total fluorescence is the sum of the two.