A platform for brain-wide imaging and reconstruction of individual neurons

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.Howard Hughes Medical InstitutePublished versio

High throughput instrument to screen fluorescent proteins under two-photon excitation

Author Posting. © Optical Society of America , 2020. This article is posted here by permission of Optical Society of America for personal use, not for redistribution. The definitive version was published in Molina, R. S., King, J., Franklin, J., Clack, N., McRaven, C., Goncharov, V., Flickinger, D., Svoboda, K., Drobizhev, M., & Hughes, T. E. High throughput instrument to screen fluorescent proteins under two-photon excitation. Biomedical Optics Express, 11(12), (2020): 7192-7203, https://doi.org/10.1364/BOE.409353.Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.National Institute of Neurological Disorders and Stroke (F31 NS108593, U01 NS094246, U24 NS109107); Howard Hughes Medical Institute

Understanding patient preference for physician attire in ambulatory clinics: a cross-sectional observational study

OBJECTIVES We explored patient perceptions regarding physician attire in different clinical contexts and resultant effects on the physician-patient relationship. SETTING The 900-bed University Hospital Zurich, Switzerland. PARTICIPANTS A convenience sample of patients receiving care in dermatology, infectious diseases and neurology ambulatory clinics of the University Hospital Zurich participated in a paper-based survey. PRIMARY AND SECONDARY OUTCOME MEASURES The survey instrument was randomised and showed photographs of male or female physicians wearing various forms of attire. On the basis of the respondents' ratings of how the physician's attire affected perceptions across five domains (knowledgeable, trustworthy, caring, approachable and comfort with the physician), a composite preference score for attire was calculated as the primary outcome. Secondary outcomes included variation in preferences by respondent characteristics and context in which care was provided. RESULTS Of 834 patient respondents (140 in dermatology, 422 in infectious diseases and 272 in neurology), 298 (36%) agreed that physician attire was important. When compared with all available choices, the combination of white scrubs with white coat was rated highest while a business suit ranked lowest. Variation in preferences and opinions for attire were noted relative to respondent demographics and the clinical setting in which the survey was administered. For example, compared with younger patients, respondents ≥65 years of age more often reported that physician dress was both important to them and influenced how happy they were with their care (p=0.047 and p=0.001, respectively). CONCLUSIONS Outpatients at a large Swiss University hospital prefer their physicians to be dressed in white scrubs with white coat. Substantial variation among respondents based on demographics, type of physician and clinical setting were observed. Healthcare systems should consider context of care when defining policies related to dress code

Automated Reconstruction of Neuronal Morphology Based on Local Geometrical and Global Structural Models

Digital reconstruction of neurons from microscope images is an important and challenging problem in neuroscience. In this paper, we propose a model-based method to tackle this problem. We first formulate a model structure, then develop an algorithm for computing it by carefully taking into account morphological characteristics of neurons, as well as the image properties under typical imaging protocols. The method has been tested on the data sets used in the DIADEM competition and produced promising results for four out of the five data sets

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Automated tracking of whiskers in videos of head fixed rodents.

We have developed software for fully automated tracking of vibrissae (whiskers) in high-speed videos (>500 Hz) of head-fixed, behaving rodents trimmed to a single row of whiskers. Performance was assessed against a manually curated dataset consisting of 1.32 million video frames comprising 4.5 million whisker traces. The current implementation detects whiskers with a recall of 99.998% and identifies individual whiskers with 99.997% accuracy. The average processing rate for these images was 8 Mpx/s/cpu (2.6 GHz Intel Core2, 2 GB RAM). This translates to 35 processed frames per second for a 640 px×352 px video of 4 whiskers. The speed and accuracy achieved enables quantitative behavioral studies where the analysis of millions of video frames is required. We used the software to analyze the evolving whisking strategies as mice learned a whisker-based detection task over the course of 6 days (8148 trials, 25 million frames) and measure the forces at the sensory follicle that most underlie haptic perception

Tracking whiskers from high-speed (500 Hz) videos during an object detection task.

<p>(A) A typical field of view. (B) Typical imaging configuration. (C–G) Automated results of tracing and linking. (C) Facial hairs and whiskers are traced in each video frame and then identified by a separate tracking step. (D) A whisker (blue) touches the pole. (E) Two whiskers (blue & green) are bent by the pole. The most posterior whisker is strongly retracted so that only a small segment is visible. (F) Tracking measures whisker orientation, such as the angle at base. (G) Tracking measures whisker shape, such as mean curvature, which can be observed over time. Changes in curvature allow the calculation of forces acting on the whisker follicle <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002591#pcbi.1002591-Birdwell1" target="_blank">[16]</a>.</p

Analysis of curvature change on contact.

<p>(A–E) The sequence of steps used to extract detailed curvature measurements. (A) Whiskers in raw video frames are automatically traced and linked, yielding (B) an identified set of curves for each frame. (C) The raw curve is fit with a 5^th-degree parametric polynomial. (D) A mask is specified to determine where the curve intersects the face. Within a small interval (1–2.5 mm path length) about an interest point chosen for high signal to noise, the raw curve is re-fit to ensure measurements are not biased by whisker shape outside the interval. This new fit is to a 2^nd-degree polynomial. The curvature at the interest point is then measured as the curvature of this 2^nd fitted curve. (E) Follicle position is estimated by extrapolating a fixed distance into the face from the mask. Similarly, curves are extrapolated, when necessary, to contact points on the pole. Trajectories for curvature (F) and the angle of the whisker at its base (G) are shown for the first contacting whisker in 10 trials grouped by whether the first contact was during a retraction (top 5) or protraction (bottom 5). Trajectories are aligned to first contact. The intervals when the whisker is in whisker-pole contact are highlighted in red. (H) Histograms of peak contact curvature change (from resting) for the first whisker-pole contact in each trial (green) and all whisker-pole contacts prior to an answer-lick (red).</p