Multifocal microscopy for functional imaging of neural systems

Significance: Rapid acquisition of large imaging volumes with microscopic resolution is an essential unmet need in biological research, especially for monitoring rapid dynamical processes such as fast activity in distributed neural systems.

Aim: We present a multifocal strategy for fast, volumetric, diffraction-limited resolution imaging over relatively large and scalable fields of view (FOV) using single-camera exposures.

Approach: Our multifocal microscopy approach leverages diffraction to image multiple focal depths simultaneously. It is based on a custom-designed diffractive optical element suited to low magnification and large FOV applications and customized prisms for chromatic correction, allowing for wide bandwidth fluorescence imaging. We integrate this system within a conventional microscope and demonstrate that our design can be used flexibly with a variety of magnification/numerical aperture (NA) objectives.

Results: We first experimentally and numerically validate this system for large FOV microscope imaging (three orders-of-magnitude larger volumes than previously shown) at resolutions compatible with cellular imaging. We then demonstrate the utility of this approach by visualizing high resolution three-dimensional (3D) distributed neural network at volume rates up to 100 Hz. These demonstrations use genetically encoded ${\mathrm{Ca}}^{2+}$ indicators to measure functional neural imaging both in vitro and in vivo. Finally, we explore its potential in other important applications, including blood flow visualization and real-time, microscopic, volumetric rendering.

Conclusions: Our study demonstrates the advantage of diffraction-based multifocal imaging techniques for 3D imaging of mm-scale objects from a single-camera exposure, with important applications in functional neural imaging and other areas benefiting from volumetric imaging.