3D Optical Reconstruction of the Nervous System of the Whole-Body Marine Invertebrates

Optical clearing of invertebrates, the number of species of which is 20 times greater than that of vertebrates, is of fundamental and applied interest for neuroscience in general. Herein, the optical clearing of invertebrates to identify their morphology and neurostructure remains unrealized as of yet. Here, we report on fast (from a few seconds to minutes) and uniform whole-body optical clearing of invertebrates (bivalves, nemertines, annelids, and anomura) of any age and thickness (up to 2 cm) possessing complicated structures and integuments. We developed the protocol unifying dimethyl sulfoxide (DMSO)-based immunostaining of the animals followed by their optical clearing with benzyl alcohol/benzyl benzoate (BABB). Confocal microspectroscopy revealed that the protocol provides an increase of the fluorescence signal by 2 orders of magnitude and decrease of the light scattering by 2 orders of magnitude, thereby accelerating the confocal bioimaging of the whole body. Moreover, by tracking the optical clearing over time with 0.3 s resolution, we revealed that the clearing process is described by the Gompertz growth function, allowing us to determine the physical mechanism of the clearing and its optical parameters. Thereby, we were able to identify in detail and to describe previously unknown neurostructures of different invertebrate animals, paving the way to discovery in neuroscience.