Breaking trade-offs: Development of fast, high-resolution, wide-field two-photon microscopes to reveal the computational principles of the brain.

Information in the brain is represented by the collective and coordinated activity of single neurons. Activity is determined by a large amount of dynamic synaptic inputs from neurons in the same and/or distant brain regions. Therefore, the simultaneous recording of single neurons across several brain regions is critical for revealing the interactions among neurons that reflect the computational principles of the brain. Recently, several wide-field two-photon (2P) microscopes equipped with sizeable objective lenses have been reported. These microscopes enable large-scale in vivo calcium imaging and have the potential to make a significant contribution to the elucidation of information-processing mechanisms in the cerebral cortex. This review discusses recent reports on wide-field 2P microscopes and describes the trade-offs encountered in developing wide-field 2P microscopes. Large-scale imaging of neural activity allows us to test hypotheses proposed in theoretical neuroscience, and to identify rare but influential neurons that have potentially significant impacts on the whole-brain system.

Fast, cell-resolution, contiguous-wide two-photon imaging to reveal functional network architectures across multi-modal cortical areas.

Fast and wide field-of-view imaging with single-cell resolution, high signal-to-noise ratio, and no optical aberrations have the potential to inspire new avenues of investigations in biology. However, such imaging is challenging because of the inevitable tradeoffs among these parameters. Here, we overcome these tradeoffs by combining a resonant scanning system, a large objective with low magnification and high numerical aperture, and highly sensitive large-aperture photodetectors. The result is a practically aberration-free, fast-scanning high optical invariant two-photon microscopy (FASHIO-2PM) that enables calcium imaging from a large network composed of ∼16,000 neurons at 7.5 Hz from a 9 mm2 contiguous image plane, including more than 10 sensory-motor and higher-order areas of the cerebral cortex in awake mice. Network analysis based on single-cell activities revealed that the brain exhibits small-world rather than scale-free behavior. The FASHIO-2PM is expected to enable studies on biological dynamics by simultaneously monitoring macroscopic activities and their compositional elements.

In-situ and real-time growth observation of high-quality protein crystals under quasi-microgravity on earth.

Precise protein structure determination provides significant information on life science research, although high-quality crystals are not easily obtained. We developed a system for producing high-quality protein crystals with high throughput. Using this system, gravity-controlled crystallization are made possible by a magnetic microgravity environment. In addition, in-situ and real-time observation and time-lapse imaging of crystal growth are feasible for over 200 solution samples independently. In this paper, we also report results of crystallization experiments for two protein samples. Crystals grown in the system exhibited magnetic orientation and showed higher and more homogeneous quality compared with the control crystals. The structural analysis reveals that making use of the magnetic microgravity during the crystallization process helps us to build a well-refined protein structure model, which has no significant structural differences with a control structure. Therefore, the system contributes to improvement in efficiency of structural analysis for "difficult" proteins, such as membrane proteins and supermolecular complexes.