Live, in vivo, imaging (GCaMP6F), Video Processing, and Analysis

AAV-PHP.S-CAG-GCaMP6F (10¹²VGs) was delivered systemically to WT C57.

3-4 weeks after infection, mice were anesthetized with 2% isoflurane on a heating pad (Kent Scientific, Torrington, CT-DCT15) with plastic sleeve covers (Kent Scientific, Torrington, CT-DCT1520P).

The abdominal cavity was surgically opened to expose the intestines.

The proximal colon was identified, and this portion was placed on top of a stack of 4-6 glass microscopy slides (depending on size of animal) (VWR, Radnor PA-Cat. No. 48300-026).

Tissue was secured onto glass slide with a biorthogonal silicon elastomer (Kwik-Sil) (World Precision Instruments, Sarasota, FL-KWIK-SIL), and a glass coverslip.

Elastomer stiffens within 1 minute of application and coverslip must be held steadily to ensure a flat imaging surface. Anesthetized mouse is placed under an upright confocal microscope (Zeiss LSM 880).

Using a 10X objective, GCaMP6F fluorescence was taken at 5Hz (1 image every 200ms).

Periods of movement of tissue and luminal contents is normal during live imaging.

Cell tracking was performed in 2D by using TrackMate ImageJ plugin (https://imagej.net/TrackMate) and fluorescence intensity was recorded from cells.

Average background was determined by taking the fluorescence of a region of interest that did not contain a cell, over the duration of the video. Background was subtracted from cell fluorescence intensities.