Three Dimensional Sub-diffractional Imaging and Optical Stimulation of Neurons

Development of new instruments results in new discoveries and opens up new research directions. In particular novel imaging and optogenetics stimulation techniques merging classical photonics with other fields have a great potential to advance medicine, biology, physics, chemistry and engineering./nHere we present the development of two novel, plasmonic-aided imaging techniques (BOM and BEAST) and describe discovery of a new activity driven plastic alteration of dendritic excitability, which was enabled by utilizing a new dynamic light modulation scheme for neural activation./nThe first technique, termed Brownian Emitter Adsorption Superresolution Technique (BEAST), is a novel single molecule super-resolution technique, which relies on stochastic adsorption of molecules to measure surface enhancement of an electromagnetic field. We used BEAST to map - for the first time - the electromagnetic field within single hotspots as small as 15nm on a metal surface with a resolution down to 1.2 nm. The hotspots are localized optical modes on the surface of noble metals exhibiting a giant enhancement effect and have attracted a broad interest, from understanding their mechanism to developing practical applications. However, characterizing these hotspots has been difficult, due to the limited resolution of current imaging techniques. BEAST improves the resolution significantly to single nanometer level, which allowed us to image the EM field inside single hotspots for the first time and discovered that the field distribution follows an exponential decay - strong experimental evidence for Anderson localization mechanism of the hotspots, which has been extensively debated over the last decades./nThe second imaging technique described here - Brownian Optical Microscopy (BOM) - is the first technique to offer true three-dimensional imaging with nano-resolution. Scanning probe microscopy (SPM), which is commonly used to image 3D topology now, offers nanoscopic resolution but the use of the tip and slow scanning speed limits its throughput and makes it unable to image high aspect ratio or cavities. On the other hand optical tomography is able to image complex three-dimensional shapes but its resolution is limited to the micron range, while electron microscopy offers higher resolution but requires imaging in vacuum. BOM does not suffer from these limitations. It is an all-optical imaging technique, which relies on Brownian motion of gold nanoparticles to sample the shape of the object, akin to scanning the sample in parallel by millions of small, freely diffusing SPM tips. In BOM an object of an arbitrary shape is placed in a solution of randomly diffusing nanoparticles, which are illuminated with an evanescent field so that the scattering intensity of resonant NPs correlates with their vertical position. We demonstrate that BOM is capable of imaging complex shapes with 30nm resolution in all three dimensions, including overhang samples, which cannot be imaged with any other technique./nFinally we use optogenetics combined with dynamic light modulation to explore plasticity of dendritic excitability. It was long believed that that plasticity, which plays a crucial role in the formation of neural circuits, is based on alteration of synaptic weights. Recent studies indicate that modulations of dendritic excitability may contribute the other part of the engram and critically impact the emergence of complex network behavior. However, a fundamental question remains whether dendritic excitability is controlled by synaptic inputs or arises independently. We used a novel optical system, which offers high spatiotemporal control over neural stimulation to decouple synaptic and non-synaptic activity and observed plasticity of local dendritic excitability which is autonomous from synaptic plasticity and arises only as a result of local activity. This persistent change in dendritic excitability arises as a result of a back propagating action potential interacting with simultaneous dendritic depolarization and triggering MEK-regulated phosphorylation of Kv4.2 by CamKII. This major discovery of activity dependent dendritic plasticity sheds new light on the role of dendrites in plasticity and may profoundly impact our fundamental understanding of neural plasticity as well as a number of neurodegenerative diseases where Kv4.2 channel deregulation is thought to play a crucial role.

Advisor: Zhang, Xiang