In Vivo Imaging of Cerebral Vessels and Amyloid Plaques in an Alzheimer’s Disease Mouse Model

Begin with an Alzheimer's disease mouse model secured on a head-mount stage. The exposed brain is covered with artificial cerebrospinal fluid, or aCSF, to maintain tissue integrity.

In the brain tissue, amyloid plaques are labeled with a blue fluorophore, while cerebral vessels are labeled with a green fluorophore.

Position the mouse under a two-photon laser microscope for deep tissue imaging.

Using the water immersion objective, locate the exposed brain.

Then, illuminate the brain with a 750-nanometer wavelength laser, which simultaneously excites blue and green fluorescent molecules.

Using fluorescence-specific filters detects the signals of amyloid plaques and cerebral vessels, enabling imaging of both structures.

Capture low-magnification images of the entire region of interest or ROI at various depths to generate a 3D map.

Then, switch to higher magnification for detailed visualization of the ROI.

This creates a high-resolution image depicting cerebral vessel networks with amyloid plaques, enabling the analysis of cerebrovascular changes during disease progression.

In this step, transfer the mouse with the head mount stage under the two-photon laser microscope. Using a 20x water immersion objective with a numerical aperture of 1.0, locate the thinned cranial window at the center of the optical field using the epifluorescent lamp. Ensure that the objective is always immersed in ACSF.

Next, start laser scanning with a mode-locked pulsed laser. Set the excitation wavelength at 750 nanometers to detect the emitted fluorescence of the Methoxy-XO4 in the blue channel, and FITC-dextran in the green channel. Acquire a low magnification stack at 0.7x numerical zoom in order to create a 3D map for the precise relocalization of the ROI at later time points.

Then acquire a mosaic of 4 high digital magnification images with a 2x numerical zoom . Using the imaging software, move the window in 200 micrometer steps to capture all regions. The depth of the stacks is typically 250 micrometers, starting from the pial surface in each image of the mosaic.