Single molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.Peer ReviewedPostprint (author's final draft

Ivermectin-activated, cation-permeable glycine receptors for the chemogenetic control of neuronal excitation

The ability to control neuronal activation is rapidly advancing our understanding of brain function and is widely viewed as having eventual therapeutic application. Although several highly effective optogenetic, optochemical genetic, and chemogenetic techniques have been developed for this purpose, new approaches may provide better solutions for addressing particular questions and would increase the number of neuronal populations that can be controlled independently. An early chemogenetic neuronal silencing method employed a glutamate receptor Cl– channel engineered for activation by 1–3 nM ivermectin. This construct has been validated in vivo. Here, we sought to develop cation-permeable ivermectin-gated receptors that were either maximally Ca2+-permeable so as to induce neuro-excitotoxic cell death or minimally Ca2+-permeable so as to depolarize neurons with minimal excitotoxic risk. Our constructs were based on the human α1 glycine receptor Cl– channel due to its high conductance, human origin, high ivermectin sensitivity (once mutated), and because pore mutations that render it permeable to Na+ alone or Na+ plus Ca2+ are well characterized. We developed a Ca2+-impermeable excitatory receptor by introducing the F207A/P-2′Δ/A-1′E/T13′V/A288G mutations and a Ca2+-permeable excitatory receptor by introducing the F207A/A-1′E/A288G mutations. The latter receptor efficiently induces cell death and strongly depolarizes neurons at nanomolar ivermectin concentrations

Axons-on-a-chip for mimicking non-disruptive diffuse axonal injury underlying traumatic brain injury

Diffuse axonal injury (DAI) is the most severe pathological feature of traumatic brain injury (TBI). However, how primary axonal injury is induced by transient mechanical impacts remains unknown, mainly due to the low temporal and spatial resolution of medical imaging approaches. Here we established an axon-on-achip (AoC) model for mimicking DAI and monitoring instant cellular responses. Integrating computational fluid dynamics and microfluidic techniques, DAI was induced by injecting a precisely controlled micro-flux in the transverse direction. The clear correlation between the flow speed of injecting flux and the severity of DAI was elucidated. We next used the AoC to investigate the instant intracellular responses underlying DAI and found that the dynamic formation of focal axonal swellings (FAS) accompanied by Ca2+ surge occurs during the flux. Surprisingly, periodic axonal cytoskeleton disruption also occurs rapidly after the flux. These instant injury responses are spatially restricted to the fluxed axon, not affecting the overall viability of the neuron in the acute stage. Compatible with high-resolution live microscopy, the AoC provides a versatile system to identify early mechanisms underlying DAI, offering a platform for screening effective treatments to alleviate TBI.Xiaorong Pan, Jie Li, Wei Li, Haofei Wang, Nela Durisic, Zhenyu Li, Yu Feng, Yifan Liu, Chun-Xia Zhao and Tong Wan

Investigating hepatitis C virus infection using super-resolution microscopy

Super-resolution microscopy (SRM) can provide a window on the nanoscale events of virus replication. Here we describe a protocol for imaging hepatitis C virus-infected cells using localization SRM. We provide details on sample preparation, immunostaining, data collection, and super-resolution image reconstruction. We have made all efforts to generalize the protocol to make it accessible to all budding super-resolution microscopists