Serial Two-Photon Tomography of the Whole Marmoset Brain for Neuroanatomical Analyses

Our research explores the structural foundation of brain functions, including those in humans. Using common marmoset as a model, we aim to better understand primate brain by identifying neural connections that are shared across species and those that are uniquely out of the primate genus. We performed a comprehensive mapping of the prefrontal projections of the marmoset brain.

Using serial two-photon tomography technique, we succeeded in highly accurate 3D reconstruction of the entire brain, which revealed robust columnar projections among the association areas. Serial two-photon tomography provides a data set for understanding neural connectivity at the whole brain scale. Our protocol not only helps improve the processing of marmoset brains, but also offers insights into handling other specimens.

To begin, take the isolated, fixed marmoset brain and incubate it with collagenase at 37 degrees Celsius for one hour. Using a cotton swab, carefully rub the brain surface to peel off the pia mater and other meninges. Use fine forceps to remove any remaining detached meninges.

To prepare the sodium borohydride buffer, dissolve 0.2 grams of sodium borohydride in 100 milliliters of 50 millimoles borate buffer, prewarmed to 40 degrees Celsius. Prepare oxidized agarose by stirring 2.25 grams of agarose and 0.21 grams of sodium periodate in 100 milliliters of phosphate buffer for two to three hours. Filter the solution using vacuum suction and wash it with three changes of phosphate buffer.

Then, resuspend the agarose in 50 milliliters of phosphate buffer. Next, completely melt the agarose in a microwave oven and allow it to cool to 60 to 65 degrees Celsius. Place the brain into a custom-made chamber and embed the brain in agarose, ensuring the agarose fills the cavity below the corpus callosum.

After solidification of the agarose gel, disassemble the chamber and immerse the agarose block in the sodium borohydride buffer overnight at four degrees Celsius. Now, using an epoxy instant mix, construct a slide glass stage by attaching four neodymium magnets. Mount the agarose block onto the stage using a strong adhesive.

Operate the whole tissue imaging system following the manufacturer's instructions. Process the entire marmoset brain coronally from the anterior to the posterior ends. Then, adjust the blade angles to ensure that the surface depths at both ends are within 10 micrometers.

To do this, search for the top surface by changing the position of the objective, using the piezo controller. Set the imaging plane to approximately 25 to 35 micrometers from the surface to compensate for dense myelination and ensure uniform laser penetration. Using a ceramic blade, cut the entire brain into 50 micrometer intervals, yielding approximately 650 slices.