Two-Photon Microscopy for In Vivo Imaging of Mouse Brain Microvasculature

Abstract

Source: Nishimura, C. et al. Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation. J. Vis. Exp. (2016).

This video demonstrates the in vivo visualization of cortical capillaries in the mouse brain using two-photon microscopy. It outlines the steps for injecting a fluorescent dye into an anesthetized mouse with a thin-skull cortical window, preparing the setup for imaging, and acquiring detailed images of brain microvascular structures for analysis.

Protocol

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

1. Injection of the Fluorescent Dye

1. Pinch the skin of an anesthetized mouse on the medial left thigh using the #5 forceps. Gently pull the skin upwards and use scissors to cut and remove the skin. Prepare dextran conjugated red-emitting rhodamine dye (70 kDa) (10 mg/kg dissolved in saline).
2. Locate the femoral vein. Using a 1 ml syringe with a 30 G needle attached, draw a previously prepared solution of a fluorescent dye appropriate for imaging to the 130 µl line of the syringe. Remove all air bubbles by drawing the dye completely into the syringe and firmly flicking the syringe wall.
   1. Bend the 30 G needle at an approximately 30 degree angle by pressing it against a hard surface bevel side up. Fill the needle with the dye and discard any excess liquid, leaving a 100 µl sample.
3. Inject the 100 µl sample of dye slowly into the femoral vein. After removing the needle, apply steady, gentle pressure to the site of injection to stop any bleeding. Allow the dye to circulate for 5 min.
4. Close the left thigh by sewing the skin together with a 4-0 suture, then carefully flip the mouse onto its stomach, and place it into the mouse headplate harness.
   

2. In Vivo Two-Photon Imaging

1. Move the surgical apparatus to the two-photon microscope, making sure to maintain continuous anesthesia levels. Place a small quantity of 0.9 % saline into the headplate reservoir and lower the microscope objective so that it comes into contact with the saline. Locate the area of interest using the brightfield-viewing objective.
2. Set the two-photon laser excitation to a wavelength appropriate for the fluorescent dye. Begin two-photon imaging using the 25x objective.
   Note that in these experiments, we used a dextran conjugated to a red-emitting rhodamine dye that was excited at a wavelength of 780 nm. A 607/36 bandpass emission filter was used to detect the fluorescence from the dye, and a 480/20 bandpass emission filter was used to detect arterial autofluorescence.
3. Locate a capillary bed on the view screen and magnify this area using optical zoom 2. Acquire images of the capillaries using the two-photon imaging software.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
T/PUMP	Gaymar Industries, Inc.	TP-500
TEC-4 Isoflurane Vaporizer	Datex Ohmeda	447
Extra Fine Bonn Scissors	Fine Science Tools	14084-08
Dumot #5 Forceps	Fine Science Tools	11295-10
Texas Red-dextran (70,000 MW, 10 mg/kg dissolved in saline)	Invitrogen	D-1830
Olympus Fluoview 1000 AOM-MPM Multiphoton Microscope	Olypmus	FV-1000 MPE
MaiTai HP DeepSee Ti:Sa laser	Spectra-Physics
Amira Software	Visage Imaging