Nov 03, 2021
Live, in vivo, imaging (GCaMP6F), Video Processing, and Analysis
- Bryan Yoo1,
- Jessica Griffiths1,
- Sarkis Mazmanian1
- 1California Institute of Technology
- Mazmanian Lab
Protocol Citation: Bryan Yoo, Jessica Griffiths, Sarkis Mazmanian 2021. Live, in vivo, imaging (GCaMP6F), Video Processing, and Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bzp7p5rn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: November 03, 2021
Last Modified: November 03, 2021
Protocol Integer ID: 54751
Keywords: analysis protocol for gcamp6f imaging, gcamp6f imaging, analysis in gut neuron, gut neuron, gcamp6f, imaging, neuron, vivo, video processing
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000375
Department of Defense
Grant ID: PD160030
National Institutes of Health
Grant ID: GM007616 and DK078938
Center for Environmental and Microbial Interactions (CEMI)
Grant ID: n/a
Heritage Medical Research Institute
Grant ID: n/a
Emerald Foundation
Grant ID: n/a
Aligning Science Across Parkinson's
Grant ID: ASAP-020495
Abstract
Protocol for GCaMP6F imaging and analysis in gut neurons used in Yoo et al 2021
Troubleshooting
Preparation
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.
Imaging
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.
Processing & Analysis
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.