The ability to visualize 3D dynamical processes like neural network activity in real time, and the complementary ability to optionally perturb these processes in a distributed spatiotemporal manner stands as one of the key ongoing challenges of modern neuroscience, and specifically the BRAIN initiative. Our group has tackled this challenge by introducing new tools, in an effort to maximize the population of neurons recorded using rapid volumetric multiphoton microscopic imaging while responding to artificial and real stimuli.
The goal of this ERC proof of concept project was specifically to advance the implementation of novel solutions for imaging a volume consisting of multiple planes simultaneously, i.e. without scanning the axial dimension, and to provide a proof of concept for their application in imaging distributed neuronal activity. The project team designed and methodically explored novel methods for scanless multiplane illumination using spatiotemporal focusing as well as complementary methods for multiplane imaging using different optical approaches. We successfully designed and constructed a prototype diffractive – refractive optical solution that can be implemented as an add-on to any wide field microscope and enables scanless, large field-of-view optical sectioning of multiple tissue planes. The prototype system was implemented as an add-on on a Nikon TE-2000U microscope, and projects a fluorescence image of a volumetric field of view of approximately 400x400x200 microns (with a 10x objective) onto a planar high-resolution and high-rate scientific CMOS camera (without any mechanical scanning).
To validate the system, we verified that the fine structures of a resolution target can be resolved across the entire volume, and that volumetric samples containing fluorescent beads can be visualized with excellent optical sectioning across the volume. Finally, we validated the ability to image volumetric bioengineered neuronal networks and to observe their dynamic activity patterns at a rate of 10 volumes/second (and potentially orders of magnitude faster), opening new vistas for high throughput 3D imaging and photo-manipulation.
