Label-free three-photon imaging of intact human cerebral organoids for tracking early events in brain development and deficits in Rett syndrome

Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (similar to 2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging.

Automated summary

This study demonstrates label-free three-photon imaging technology for assessing early events of early human brain development in whole, uncleared intact organoids. By imaging cerebral organoids generated from Rett Syndrome patients, the researchers found defects in the volumetric structure of the ventricular zone in mutant organoids compared to control organoids. Live imaging of organoids also revealed that mutant radially migrating neurons had shorter migration distances and slower migration speeds.