Speed limits of structured illumination microscopy

A theoretical framework for widefield structured illumination microscopy (SIM) reconstruction from fewer than the commonly used nine raw frame acquisitions is introduced and applied in silico and in vitro. The proposed scheme avoids the recording of redundant spatial frequency components, which was necessary in previous SIM algorithms. This allows for gentler superresolution imaging at faster speeds. A doubling of frame rates is possible solely via changes in the computational reconstruction procedure. Furthermore, we explore numerically the effect of the sample movement on the reconstruction quality and the number of raw frames recordable. Our results show that there exists a limit above which deconvolution microscopy becomes superior to SIM.Engineering and Physical Sciences Research Council (EPSRC) (EP/H018301/1); Medical Research Council (MRC) (MR/K015850/1, MR/K02292X/1); Wellcome Trust (089703/Z/09/Z)

RNA Docking and Local Translation Regulate Site-Specific Axon Remodeling In Vivo

Nascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses, but it is unknown whether LPS influences these responses in vivo. Using high-resolution live imaging, we examined the spatiotemporal dynamics of RNA and LPS in retinal axons during arborization in vivo. Endogenous RNA tracking reveals that RNA granules dock at sites of branch emergence and invade stabilized branches. Live translation reporter analysis reveals that de novo ß-actin hotspots colocalize with docked RNA granules at the bases and tips of new branches. Inhibition of axonal ß-actin mRNA translation disrupts arbor dynamics primarily by reducing new branch emergence and leads to impoverished terminal arbors. The results demonstrate a requirement for LPS in building arbor complexity and suggest a key role for pre-synaptic LPS in assembling neural circuits.This work was supported by Cambridge Trust, Croucher Foundation, Sir Edward Youde Memorial Fund (H.H.-W.W.), Gates Cambridge (J.Q.L.), Fundac¸ a˜ o para a Cieˆ ncia e Tecnologia (C.M.R.), Wellcome Trust Senior Investigator Award (100329/Z/ 12/Z) (W.A.H.), EPSRC Grant (EP/H018301/1), MRC Grant (MR/K015850/1 and MR/K02292X/1), Wellcome Trust (089703/Z/09/Z) (C.F.K.), Wellcome Trust Programme Grant (085314/Z/08/Z), and ERC Advanced Grant (322817) (C.E.H.)

Single Molecule Translation Imaging Visualizes the Dynamics of Local β-Actin Synthesis in Retinal Axons

Local mRNA translation occurs in growing axons enabling precise control of the proteome in response to signals. To measure quantitatively the spatiotemporal dynamics of protein synthesis in growth cones, we further developed a technique for single molecule translation imaging (SMTI). We report that Netrin-1 triggers a burst of β-actin synthesis at multiple non-repetitive sites, particularly in the periphery. The response is remarkably rapid starting within 20 seconds of cue application.This work was supported by grants from the Leverhulme Trust, the Engineering and Physical Sciences Research Council, UK (grant EP/H018301/1), the Medical Research Council (grant MR/K015850/1, and MR/K02292X/1), the Wellcome Trust (089703/Z/09/Z) (C.F.K.), Sir Edward Youde Memorial Fund, Croucher Foundation, Cambridge Trust (H.H.W.) Gates Cambridge Scholarship (J.Q.L.), Wellcome Trust Studentship (V.U.), European Research Council Advanced Grant (322817), the Wellcome Trust (085314/Z/08/Z) (C.E.H.)

Batch calculation of mean and SD evolution

This Matlab code enables reproduction of the analysis results used in Figure 7 in Wong et al. (2017). The script takes drift-corrected fluorescence recovery after photobleaching time-series as well as masks of the axonal arbour (divided into shaft and branch masks) as inputs and calculates mean and standard deviation of time-resolved recovery within the branches and shafts as well as their relative recoveries for different experimental conditions