One-photon calcium imaging in C. elegans with novel acousto-optic technique

One-photon (1P) imaging and optical stimulation are two powerful tools widely used to assess and monitor the behaviour of individual and multiple neurons in in-vivo and ex-vivo experiments. However, standard 1P microscopes are limited by diffraction and scattering, which reduce the maximum optical depth achievable to few hundreds of micrometres deep into brain tissues. To overcome this issue, implantable waveguides and probes (i.e. optical fibres) are commonly used, but they are highly-invasive and may lead to damage the surrounding tissue. In this work, it has been developed a customized 1P setup fully compatible with an innovative acousto-optic technology based on an ultrasonic phased-array, which allows to sculpt a non-invasive virtual waveguide within the tissue, by locally changing its refractive index. Indeed, this technology is capable to deliver and confine light in tissue phantoms with optical thickness (OT) up to 18.8 Mean-Free-Path (MFP) and to perform imaging in turbid media (OT up to 52 MFP). Furthermore, not all laboratories are allowed to use model animals such as monkeys or mice in their experiments, because adequate facilities and surgical skills are needed. Moreover, ex-vivo experiments are complex and time-limited, since specimens need special treatments to be kept alive and, anyway, after a couple of hours they are no longer able to be used, as clinically dead. For this purpose, here is presented a new concept, cost-effective brain tissue phantom, which exploits living transgenic Caenorhabditis elegans (C. elegans) to replicate the structure of a living brain tissue. This can be easily used as proof of concept demonstration of new applications in experiments like electrophysiology, or optogenetics. The setup has been tested by carrying out experiments on fixed mouse brain slices with green fluorescent protein (GFP) and on living GCaMP6s C. elegans.