Deep-Tissue Imaging of a Zebrafish Brain Using a Three-Photon Fluorescence Microscope

Place a Petri dish containing an anesthetized zebrafish with tubing inserted into its mouth to deliver oxygenated water under the low-magnification objective lens of the three-photon microscope.

The zebrafish's brain contains genetically modified neurons expressing fluorescently labeled proteins for deep-tissue visualization during imaging.

Illuminate the dish using an LED light source, and adjust the visualization parameters for image clarity.

Lower the objective lens until the zebrafish is visible, centering its head in the field of view.

Replace the low-magnification lens with a high-resolution objective for three-photon imaging.

Carefully lower the objective until the head is visible without physical contact, and set the axis values to zero.

Turn off the LED light source and close the dark curtain to minimize background noise.

Set the imaging parameters and perform three-photon imaging, where longer wavelengths enable deep-tissue three-photon excitation.

Upon excitation, neuronal fluorophores emit fluorescence, enabling real-time visualization of neurons at various depths.

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.