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Mechanistic understanding of how the brain gives rise to complex behavioral and cognitive functions is one of science's grand challenges. The technical challenges that we face as we attempt to gain a systems-level understanding of the brain are manifold. The brain's structural complexity requires us to push the limit of imaging resolution and depth, while being able to cover large areas, resulting in enormous data acquisition and processing needs. Furthermore, it is necessary to detect functional activities and 'map' them onto the structural features. The functional activity occurs at multiple levels, using electrical and chemical signals. Certain electrical signals are only decipherable with sub-millisecond timescale resolution, while other modes of signals occur in minutes to hours. For these reasons, there is a wide consensus that new tools are necessary to undertake this daunting task. Optical techniques, due to their versatile and scalable nature, have great potentials to answer these challenges. Optical microscopy can now image beyond the diffraction limit, record multiple types of brain activity, and trace structural features across large areas of tissue. Genetically encoded molecular tools opened doors to controlling and detecting neural activity using light in specific cell types within the intact brain. Novel sample preparation methods that reduce light scattering have been developed, allowing whole brain imaging in rodent models. Adaptive optical methods have the potential to resolve images from deep brain regions. In this roadmap article, we showcase a few major advances in this area, survey the current challenges, and identify potential future needs that may be used as a guideline for the next steps to be taken.

Structured illumination microscopies achieve optical sectioning via differential modulation of in-focus and out-of-focus contributions to an image. Multiple wide-field camera images are analyzed to recreate an optical section. The requirement for multiple camera frames per image entails a loss of temporal resolution compared to conventional wide-field imaging. Here we describe a computational structured illumination imaging scheme, compressed Hadamard imaging, which achieves simultaneously high spatial and temporal resolution for optical sectioning of 3D samples with low-rank dynamics (e.g. neurons labeled with fluorescent activity reporters). We validate the technique with numerical simulations, and then illustrate with wide-area optically sectioned recordings of membrane voltage dynamics in mouse neurons in an acute brain slice and of calcium dynamics in zebrafish brain in vivo.