Advanced Optical Techniques to Explore Brain Structure and Function

Abstract

Understanding brain structure and function, and the complex relationships between them, is one of the grand challenges of contemporary sciences. Thanks to their°exibility, optical techniques could be the key to explore this complex network. In this manuscript, we brie°y review recent advancements in optical methods applied to three main issues: anatomy, plasticity and functionality. We describe novel implementations of light-sheet microscopy to resolve neuronal anatomy in whole¯xed brains with cellular resolution. Moving to living samples, we show how real-time dynamics of brain rewiring can be visualized through two-photon microscopy with the spatial resolution of single synaptic contacts. The plasticity of the injured brain can also be dissected through cutting-edge optical methods that speci¯cally ablate single neuronal processes. Finally, we report how nonlinear microscopy in combination with novel voltage sensitive dyes allow optical registrations of action potential across a population of neurons opening promising prospective in understanding brain functionality. The knowledge acquired from these complementary optical methods may provide a deeper comprehension of the brain and of its unique features.

Clearing of fixed tissue: a review from a microscopist’s perspective

“…7 A prominent reason for the flourishing of these techniques is the concurrent development of high-throughput microscopy imaging systems, which allow the reconstruction of large specimens with micrometric resolution. 36 Nonetheless, the landscape of clearing technologies is often depicted just in chemical terms, without taking into proper account the imaging methods to which clearing protocols should be eventually coupled. Here, we tried to describe and classify existing techniques from a microscopist's perspective, highlighting the fact that there is no "ultimate" clearing protocol, surmounting all the others in all applications.…”

“…The sequential acquisition scheme usually employed in confocal and two-photon microscopy limits imaging speed, 36 restricting the common use of these methods only portions of entire organs. 19 To afford full reconstruction of whole mouse brains, significant engineering efforts are required to keep system stability over long (several weeks) imaging times.…”

Plastic embedding immunolabeled large-volume samples for three-dimensional high-resolution imaging

“…Specifically, the iDISCO immunostaining technique [5] shows good antibody compatibility and can better implement IF-labeled large-volume samples. On the other hand, with the advances in optical imaging technology, the combination of tissue optical clearing, optical projection tomography (OPT), and light sheet imaging technology enables 3D imaging of IF-labeled intact tissue [5][6][7][8][9][10][11][12]. However, acquiring more detailed information of endogenous molecule distribution in large-volume biological tissue remains challenging, largely owing to the finite resolution of OPT and light sheet technology [12,13].…”

Dual-slit confocal light sheet microscopy for in vivo whole-brain imaging of zebrafish

“…Electrophysiological methods can record activities in high temporal resolution, but the quantity of neurons these techniques recorded is limited by the density of electrodes. Thanks to the advent of calcium fluorescent label methods, in vivo imaging approaches such as laser scanning confocal microscopy (LSCM) [6], multiphoton microscopy (MPM) [7,8] and light sheet based microscopy (LSM) [9,10] have been developed to better visualize neuron's calcium activities in living animals [2,11].…”