Optimized Volumetric Live Imaging with Light Field Microscopy and Selective Volume Illumination

Many important biological processes, e.g. brain activity, depend on the tight spatio-temporal coordination between cells across tissue-scale 3D space. Thus a cellular-level understanding of these processes could greatly benefit from a volumetric live imaging modality, where a single snapshot could capture spatial information over a tissue-scale axially-extended volume, while maintaining single- or few-cell resolution. Such capability has been recently demonstrated with Light Field Microscopy (LFM), where the light field coming from the sample space is recorded in a single 2D image, capturing information from the 3D volume extended above and below the native focal plane, albeit at reduced spatial resolution due to the diffraction limit. Computational reconstruction is used to generate the 3D images of the sample from the 2D light field images. Up until now, LFM has been done with widefield illumination, essentially lighting up the entirety of the sample, even though only a part of the sample is meaningfully captured in the reconstruction. Thus, widefield illumination introduces extraneous background and noise, degrading the contrast and accuracy of the final reconstructed images. Here, we implement Selective Volume Illumination (SVI) to LFM, illuminating only the volume of interest, thus significantly reducing the background and providing higher contrast and accuracy for the light field image reconstruction. We demonstrate SVI-LFM with both 1-photon and 2-photon excitation, the latter providing higher penetration depth in scattering tissue. Additionally, our implementation allows recording a high resolution, optically-sectioned 3D image of the same sample, in addition to the light-field-reconstructed volumetric, but low-resolution, image. Having both the high-resolution/low-speed, and low-resolution/high-speed image data of the same sample facilitates optimized observation of dynamical processes, and provides the potential for using the high-resolution image data to help speed up and constrain the reconstruction of the light field data.