Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

The protocol illustrates how to perform in vivo deep-tissue three-photon microscopy in mouse brain and adult zebrafish. Two important model systems in life sciences. Long-wavelength three-photon microscopy enables in vivo high-spatial resolution imaging of intact tissues or organs at depths that are inaccessible by two-photon microscopy.

This technique can help us understand the underlying mechanisms of neurological diseases, such as Alzheimer's and autism, where a complete understanding of how the disease affects the brain is lacking. Demonstrating the mouse brain imaging procedure will be Kibaek Choe, the post-doctoral researcher from our laboratory and demonstrating the zebrafish brain imaging procedure will be Kristine Kolkman, a research associate from the Fetcho Research Laboratory. To begin, turn on the laser and set the center wavelength of the idler output of the non-collinear optical parametric amplifier at 1, 300 or 1, 700 nanometers, then place pulse compressors on the light path to pre-chirp the femtosecond laser and optimize the pulse duration for three-photon imaging.

Set the angle between the silicon plate and laser path at Brewster's angle to maximize the laser transmittance, then rotate the silicon plate to achieve Brewster's angle by minimizing the reflection. Next, place flipper mirrors to conveniently switch between the 1, 300 and 1, 700-nanometer beam lines. Place a half waveplate mounted on a rotation stage and a polarizing beam splitter to control the intensity of the laser, then place a beam blocker in the reflection path of the beam splitter.

Prepare a Petri dish with 0.5 centimeters of 2%high-melting point agar. Once the agar has cooled and solidified, cut a rectangular hole in the agar longer and slightly wider than the fish. Using wax, attach thin tubing to the Petri dish with one end in the rectangle and a larger-diameter tubing to the edge of the Petri dish.

Next, anesthetize the fish by placing it in a 0.2 milligrams per milliliter tricaine solution, then place the anesthetized fish on its side on a wet sponge, then using a microsyringe, retro-orbitally inject 3 microliters of pancuronium bromide to paralyze the fish and briefly place it in Hank's solution to ensure it is fully paralyzed. Next, place the fish dorsal-side up in the Petri dish with the head toward the tubing, then using forceps, gently open the fish's mouth and slide the tubing into the mouth. Gently slide the fish toward the tubing so that the tubing will be at the back of the fish's mouth.

Quickly, but gently, dry the agar around the fish and remove the water on top of the fish. Dip a small piece of laboratory tissue into laboratory glue and put the tissue onto the agar on both sides of the fish and over the fish's back caudal to the gills. Next, anesthetize the fish's skin by placing a small drop of bupivacaine directly on the surface of the head, then bring the Petri dish with the fish to the microscope.

Fill the dish with fish facility water and connect to the tubing to a water pump to pump system water into the fish's mouth. Ensure that the water is oxygenated with a bubbler and warmed to 30 degrees Celsius with an aquarium heater. For imaging, place a low-magnification objective on the three-photon microscope, then place the Petri dish containing the fish and the tubes under the microscope and use an LED light source to illuminate the Petri dish.

From the image acquisition software, open the Camera mode, click Live, choose channel A on the right side of the screen and adjust the histogram settings to see the image clearly. After setting the motor setting on the motor controller to base, lower the objective until the fish is visible. Place the center of the fish head at the center of the field of view, then move the objective up and away from the fish's head.

Replace the low-magnification objective lens with the high-numerical aperture objective lens for three-photon imaging, then move it close towards the fish's head. Set the axis values of all motors to zero. Slowly lower the objective lens, ensuring that the objective does not contact the head physically.

In the CCD camera software, stop moving the objective when the top of the head is visible and set the X, Y, and Z locations to zero. Turn off the LED light source and close the dark curtain around the system, then set the imaging acquisition software to multiphoton GG mode for three-photon imaging and set the power under the objective lens to less than one milliwatt. Click on the Live button in the image acquisition software.

Open PMT channels and adjust the PMT gain and background level as needed. Slowly move up the objective lens to locate the surface of the head by monitoring the THG channel from the large blood vessels and the window glass surface. After moving the objective lens close to the window, apply water between the objective and the cranial window, then set axis values of all motors to zero.

Click on the Live button in the image acquisition software. Open PMT channels and adjust the PMT gain and background level as needed, then slowly move up the objective lens to locate the surface of the cover slip by monitoring the THG channel from the large blood vessels and the window glass surface. Adjust the window orientation if needed, then zero the motors to define the surface of the brain.

Perform imaging and adjust the power level according to the imaging depth. High-resolution, non-invasive, and deep imaging of genetically-labeled neurons in the adult zebrafish brain is achieved using three-photon microscopy. The distribution of cell layers in the optic tectum and cerebellum can also be observed.

The camera feature of the microscope was used to locate the fish. The skull is seen in the THG channel, which helps navigate the brain and find the top surface. Neurons are distinguishable with a high signal-to-background ratio deep in the adult brain.

Multicolor three-photon images of GCaMP6s-labeled neurons and Texas Red-labeled blood vessels together with THG signals in the adult mouse brain are shown here. With setup optimization, high-contrast images were successfully obtained down to 1.2 millimeters from the brain surface in the CA1 hippocampus region. Calcium activity traces of GCaMP6s-labeled neurons at a depth of 750 micrometers for a 10-minute recording session showed high recording fidelity.

This setup can help visualize neuronal activity to understand brain function. It can also be used to track cell movement within biological tissue to understand various biological systems. Additionally, blood flow speed can also be measured to monitor the animal's health.