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 contain