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1310 Janelia Publications

Showing 1-10 of 1310 results
06/01/18 | Adaptive optical microscopy for neurobiology.
Rodriguez C, Ji N
Current Opinion in Neurobiology. 2018 Jun;50:83-91. doi: 10.1016/j.conb.2018.01.011


  • Biological specimens introduce wavefront aberrations and deteriorate the image quality of optical microscopy.
  • Adaptive optics is used in optical microscopy to recover ideal imaging performance.
  • Adaptive optical imaging improves structural imaging of neurons, allowing for synaptic-level resolution at depth.
  • Adaptive optical imaging leads to a more accurate characterization of the functional properties of neurons.

With the ability to correct for the aberrations introduced by biological specimens, adaptive optics—a method originally developed for astronomical telescopes—has been applied to optical microscopy to recover diffraction-limited imaging performance deep within living tissue. In particular, this technology has been used to improve image quality and provide a more accurate characterization of both structure and function of neurons in a variety of living organisms. Among its many highlights, adaptive optical microscopy has made it possible to image large volumes with diffraction-limited resolution in zebrafish larval brains, to resolve dendritic spines over 600μm deep in the mouse brain, and to more accurately characterize the orientation tuning properties of thalamic boutons in the primary visual cortex of awake mice.

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03/18/18 | Model-free quantification and visualization of colocalization in fluorescence images.
Taylor AB, Ioannou MS, Aaron J, Chew T
Cytometry. Part A : the journal of the International Society for Analytical Cytology. 2018 Mar 13:. doi: 10.1002/cyto.a.23356

The spatial association between fluorescently tagged biomolecules in situ provides valuable insight into their biological relationship. Within the limits of diffraction, such association can be measured using either Pearson's Correlation Coefficient (PCC) or Spearman's Rank Coefficient (SRC), which are designed to measure linear and monotonic correlations, respectively. However, the relationship between real biological signals is often more complex than these measures assume, rendering their results difficult to interpret. Here, we have adapted methods from the field of information theory to measure the association between two probes' concentrations based on their statistical dependence. Our approach is mathematically more general than PCC or SRC, making no assumptions about the type of relationship between the probes. We show that when applied to biological images, our measures provide more intuitive results that are also more robust to outliers and the presence of multiple relationships than PCC or SRC. We also devise a display technique to highlight regions in the input images where the probes' association is higher versus lower. We expect that our methods will allow biologists to more accurately and robustly quantify and visualize the association between two probes in a pair of fluorescence images. © 2018 International Society for Advancement of Cytometry.

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03/12/18 | Nociceptive interneurons control modular motor pathways to promote escape behavior in.
Burgos A, Honjo K, Ohyama T, Qian CS, Shin GJ, Gohl DM, Silies M, Tracey WD, Zlatic M, Cardona A, Grueber WB
eLife. 2018 Mar 12;7:. doi: 10.7554/eLife.26016

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. Inlarvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. We identified a population of interneurons in the nerve cord of, termed Down-and-Back (DnB) neurons, that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior.

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03/07/18 | cisTEM, User-friendly software for single-particle image processing.
Grant T, Rohou A, Grigorieff N
eLife. 2018 Mar 07;7:. doi: 10.7554/eLife.35383

We have developed new open-source software calledTEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging.TEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It implements a full processing pipeline including movie processing, image defocus determination, automatic particle picking, 2D classification, ab-initio 3D map generation from random parameters, 3D classification, and high-resolution refinement and reconstruction. Some of these steps implement newly-developed algorithms; others were adapted from previously published algorithms. The software is optimized to enable processing of typical datasets (2000 micrographs, 200k - 300k particles) on a high-end, CPU-based workstation in half a day or less, comparable to GPU-accelerated processing. Jobs can also be scheduled on large computer clusters using flexible run profiles that can be adapted for most computing environments.TEM is available for download from

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03/01/18 | From electron crystallography of 2D crystals to MicroED of 3D crystals.
Martynowycz MW, Gonen T
Current Opinion in Colloid & Interface Science . 2018 Mar;34:9-16. doi: 10.1016/j.cocis.2018.01.010

Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.

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02/26/18 | A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.
Piatkevich KD, Jung EE, Straub C, Linghu C, Park D, Suk H, Hochbaum DR, Goodwin D, Pnevmatikakis E, Pak N, Kawashima T, Yang C, Rhoades JL, Shemesh O, Asano S, Yoon Y, Freifeld L, Saulnier JL, Riegler C, Engert F, Hughes T, Drobizhev M, Szabo B, Ahrens MB, Flavell SW, Sabatini BL, Boyden ES
Nature Chemical Biology. 2018 Feb 26:. doi: 10.1038/s41589-018-0004-9

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

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02/24/18 | Integrative whole-brain neuroscience in larval zebrafish.
Vanwalleghem GC, Ahrens MB, Scott EK
Current Opinion in Neurobiology. 2018 Feb 24;50:136-145. doi: 10.1016/j.conb.2018.02.004

Due to their small size and transparency, zebrafish larvae are amenable to a range of fluorescence microscopy techniques. With the development of sensitive genetically encoded calcium indicators, this has extended to the whole-brain imaging of neural activity with cellular resolution. This technique has been used to study brain-wide population dynamics accompanying sensory processing and sensorimotor transformations, and has spurred the development of innovative closed-loop behavioral paradigms in which stimulus-response relationships can be studied. More recently, microscopes have been developed that allow whole-brain calcium imaging in freely swimming and behaving larvae. In this review, we highlight the technologies underlying whole-brain functional imaging in zebrafish, provide examples of the sensory and motor processes that have been studied with this technique, and discuss the need to merge data from whole-brain functional imaging studies with neurochemical and anatomical information to develop holistic models of functional neural circuits.

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02/23/18 | BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.
McArthur K, Whitehead LW, Heddleston JM, Li L, Padman BS, Oorschot V, Geoghegan ND, Chappaz S, Davidson S, San Chin H, Lane RM, Dramicanin M, Saunders TL, Sugiana C, Lessene R, Osellame LD, Chew T, Dewson G, Lazarou M, Ramm G, Lessene G, Ryan MT, Rogers KL, van Delft MF, Kile BT
Science (New York, N.Y.). 2018 02 23;359(6378):. doi: 10.1126/science.aao6047

Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.

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02/20/18 | VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis.
Ott C, Nachmias D, Adar S, Jarnik M, Sherman S, Birnbaum RY, Lippincott-Schwartz J, Elia N
Scientific Reports. 2018 Feb 20;8(1):3353. doi: 10.1038/s41598-018-21491-x

The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4, which can't hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer's vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.

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02/19/18 | Single excitatory axons form clustered synapses onto CA1 pyramidal cell dendrites
Bloss EB, Cembrowski MS, Karsh B, Colonell J, Fetter RD, Spruston N
Nature. 2018:. doi: 10.1038/s41593-018-0084-6

CA1 pyramidal neurons are a major output of the hippocampus and encode features of experience that constitute episodic memories. Feature-selective firing of these neurons results from the dendritic integration of inputs from multiple brain regions. While it is known that synchronous activation of spatially clustered inputs can contribute to firing through the generation of dendritic spikes, there is no established mechanism for spatiotemporal synaptic clustering. Here we show that single presynaptic axons form multiple, spatially clustered inputs onto the distal, but not proximal, dendrites of CA1 pyramidal neurons. These compound connections exhibit ultrastructural features indicative of strong synapses and occur much more commonly in entorhinal than in thalamic afferents. Computational simulations revealed that compound connections depolarize dendrites in a biophysically efficient manner, owing to their inherent spatiotemporal clustering. Our results suggest that distinct afferent projections use different connectivity motifs that differentially contribute to dendritic integration.

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