Label-free three-photon imaging of intact human cerebral organoids: 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 (~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 of 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.