Intravital Imaging: A Method for Cellular Level Observation of the Mouse Pancreas Through a Stabilized Imaging Window

Overview

In this video, we describe the intravital imaging of a mouse pancreas through a stabilized window for long-term visualization of cellular dynamics.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Intravital imaging

1. Turn on the intravital microscope including the laser power.
2. Turn on the heating pad and set the homeothermic regulation to 37 °C.
   NOTE: Alternatively, use a passive heating pad or lamp with frequent control if there is no homeothermic regulation.
3. Take a mouse with pre-implanted pancreatic window. Perform intramuscular anesthesia with a mixture of tiletamine/zolazepam (30 mg/kg) and xylazine (10 mg/kg).
   NOTE: Use of tiletamine/zolazepam is recommended instead of ketamine because of its adverse effect of hyperglycemia. Optimal anesthesia should be selected for the purpose of the experiments.
4. Insert a vascular catheter for the injection.
   1. Apply pressure on the proximal side of the tail with the index and third finger as an alternative to a tourniquet application. Heat the tail with a lamp if needed.
   2. Sterilize the tail vein with a 70% ethanol spray.
   3. Insert a 30 G catheter into the lateral tail vein. Regurgitation of blood will be visualized in the PE10 tube.
   4. Apply a silk tape on the catheter to stabilize it.
   5. Inject FITC/TMR dextran or other fluorescent probes (25 µg of anti-CD31 conjugated with Alexa 647), as appropriate, according to the combination of fluorescent probes.
      NOTE: For fluorescent conjugated antibody probes, inject 2 h before the imaging session.
5. Transfer the mouse from the surgical platform to the imaging stage.
6. Insert a rectal probe to automatically control the body temperature with the homeothermic heating pad system.
7. Insert the pancreatic imaging window into the window holder prepared during the intravital microscopy setup (Figure 1). For an inverted microscope, a window holder might not be required.
8. Perform intravital imaging.
   1. For imaging the pancreas, start with a low magnification objective lens (e.g., 4x) for scanning the whole view of the pancreas in the pancreatic imaging window (recommended field of view: 2500 x 2500 µm).
   2. After determination of the region of interest, switch to higher magnification objective lens (20x or 40x) to perform the cellular level imaging (recommended field of view: 500 x 500 µm or 250 x 250 µm). In this experiment, the lateral and axial resolution was approximately 0.5 µm and 3 µm, respectively.
   3. Perform z-stack or time-lapse imaging to observe the 3D structure or cellular-level dynamics, such as cell migration.
      NOTE: For imaging the fluorescent protein expressing cells of transgenic animals (MIP-GFP), 30 s of intermittent 488 nm laser exposure with power up to 0.43 mW was tolerable without noticeable photobleaching or tissue damage. For imaging the fluorescent proteins labeled with Alexa 647, the 640 nm laser power up to 0.17 mW was tolerable without noticeable photobleaching or tissue damage. Prolonged excitation laser exposure with a power above this setting may lead to photobleaching or tissue damage by phototoxicity. Adjust the adequate gain and power to appropriately image the region of interest. Detailed setting of parameters in intravital microscopy must be individualized for each intravital microscopy prepared in the institute.

Representative Results

Figure 1: Photograph of the implementation of the pancreatic intravital imaging window. A pancreatic intravital imaging window is implanted in the mouse in the XYZ translational stage and the imaging chamber holder attached to the tilting mount is connected to the pancreatic imaging window. Copyright 2020 Korean Diabetes Association from Diabetes Metab J. 2020 44:1:193-198. Reprinted with permission from The Korean Diabetes Association.

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 647 Succinimidyl Esters (NHS esters)	Invitrogen	A20006	Fluorescent probe for conjugate with antibody
BD Intramedic polyethylene tubing	BD Biosciences	427401	PE10 catheter for connection with needle
C57BL/6N	OrientBio	C57BL/6N	C57BL/6N
Cover glasses circular	Marienfeld	0111520	Cover glass for pancreatic imaging window
FITC Dextran 2MDa	Merck (Former Sigma Aldrich)	FD200S	For vessel identification
IMARIS 8.1	Bitplane	IMARIS	Image processing
Intravital Microscopy	IVIM tech	IVM-C	Intravital Microscopy
MIP-GFP	The Jackson Laboratory	006864	B6.Cg-Tg(Ins1-EGFP)1Hara/J
Pancreatic imaging window	Geumto Engineering	Custom order	Pancreatic imaging window - custom order
Physiosuite	Kent Scientific	PS-02	Homeothermic temperature controller
TMR Dextran 65-85kDa	Merck (Former Sigma Aldrich)	T1162	For vessel identification
Window holder	Geumto Engineering	Custom order	Window holder - custom order
Zoletil	Virbac	Zoletil 100	Anesthetic agent
Rompun	Bayer	Rompun	Anesthetic agent