Optical volumetric projection with large NA objectives for fast high-resolution 3D imaging of neural signals

One critical challenge in studying neural circuits of freely behaving model organisms is to record neural signals distributed within the whole brain, yet simultaneously maintaining cellular resolution. However, due to the dense packing of neuron cells in animal brains, high numerical aperture (NA) objectives are often required to differentiate neighboring neurons with the consequent need for axial scanning for whole brain imaging. Extending the depth of focus (EDoF) will be beneficial for fast 3D imaging of those neurons. However, current EDoF-enabled microscopes are primarily based on objectives with small NAs (≤0.3 ) such that the paraxial approximation can be applied. In this paper, we started from a nonparaxial approximation of the defocus aberration and derived a new phase mask that was appropriate for large NA microscopic systems. We validated the performance experimentally with a spatial light modulator (SLM) to create the designed phase mask. The performance was tested on different samples such as multilayered fluorescence beads and thick brain tissues, as well as with different objectives. Results confirmed that our design has extended the depth of focus about 10 fold and the image quality is much higher than those based on the most common EDoF method, the cubic phase method, popularly used to generate Airy beams. Meanwhile, our phase mask is rotationally symmetric and easy to fabricate. We fabricated one such phase plate and tested it on the pan-neuronal labeled Caenorhabditis elegans (C.elegans). The imaging performance demonstrated that we can capture all neurons in the whole brain with one snapshot and with cellular resolution, while the imaging speed is increased about 3 fold compared to the system using SLM. Thus we have shown that our method can not only provide the required imaging speed and resolution for studying neural activities in model animals, but also can be implemented as a low-cost, add-on module that can immediately augment existing fluorescence microscopes with only minor system modifications, and yielding substantially higher photon efficiency than SLM-based methods.