ABSTRACT
BACKGROUND: This study aimed to clarify the variability in the measurements of stress sonography of the ankle and determine the effects of examiner experience on the measurements. METHODS: Twenty examiners (10 experienced and 10 beginners) were included in the study. Each examiner performed stress ultrasonography on a patient with a chronic anterior talofibular ligament injury and a patient with an intact ligament using the reverse anterior drawer method. Changes in ligament length before versus after stress were determined. The same 20 examiners performed ultrasonography on two other patients with an injured or intact ATFL using the anterior drawer method. The length change values and variance were compared between the groups using t-tests and F-tests. RESULTS: Using the reverse anterior drawer method, the change in the anterior talofibular ligament length was 3.3 mm (range, 2.2-4.8 mm) in the experienced group and 2.7 mm (0.0-4.1 mm) in the beginner group for the ligament injured patient. The length changes for the patient with intact anterior talofibular ligament were 0.5 mm (0.1-0.9 mm) and 0.4 mm (-0.1-1.5 mm) in the experienced and beginner groups, respectively. There were no significant intergroup differences in measurement amount (P = 0.37) or variance (P = 0.72). Similarly, using the anterior drawer method, no significant differences between the groups were found in measurement amount or variance. CONCLUSION: The quantitative evaluation of stress sonography of the ankle was variable regardless of examiner experience or stress method, particularly in patients with an anterior talofibular ligament injury. The amount of variability appeared to be unacceptably large for clinical application. Our study results highlight the need for technical standardization.
Subject(s)
Ankle Injuries , Joint Instability , Lateral Ligament, Ankle , Humans , Ankle , Ankle Injuries/diagnostic imaging , Ankle Joint/diagnostic imaging , Lateral Ligament, Ankle/diagnostic imaging , Lateral Ligament, Ankle/injuries , Ultrasonography/methodsABSTRACT
Significance: HiLo microscopy synthesizes an optically sectioned image from two images, one obtained with uniform and another with patterned illumination, such as laser speckle. Speckle-based HiLo has the advantage of being robust to aberrations but is susceptible to residual speckle noise that is difficult to control. We present a computational method to reduce this residual noise without undermining resolution. In addition, we improve the versatility of HiLo microscopy by enabling simultaneous multiplane imaging (here nine planes). Aim: Our goal is to perform fast, high-contrast, multiplane imaging with a conventional camera-based fluorescence microscope. Approach: Multiplane HiLo imaging is achieved with the use of a single camera and z-splitter prism. Speckle noise reduction is based on the application of a non-local means (NLM) denoising method to perform ensemble averaging of speckle grains. Results: We demonstrate the capabilities of multiplane HiLo with NLM denoising both with synthesized data and by imaging cardiac and brain activity in zebrafish larvae at 40 Hz frame rates. Conclusions: Multiplane HiLo microscopy aided by NLM denoising provides a simple tool for fast optically sectioned volumetric imaging that can be of general utility for fluorescence imaging applications.
Subject(s)
Lighting , Microscopy , Animals , Zebrafish , Light , LasersABSTRACT
There has been recent interest in the development of fluorescence microscopes that provide high-speed volumetric imaging for life-science applications. For example, multi-z confocal microscopy enables simultaneous optically-sectioned imaging at multiple depths over relatively large fields of view. However, to date, multi-z microscopy has been hampered by limited spatial resolution owing to its initial design. Here we present a variant of multi-z microscopy that recovers the full spatial resolution of a conventional confocal microscope while retaining the simplicity and ease of use of our initial design. By introducing a diffractive optical element in the illumination path of our microscope, we engineer the excitation beam into multiple tightly focused spots that are conjugated to axially distributed confocal pinholes. We discuss the performance of this multi-z microscope in terms of resolution and detectability and demonstrate its versatility by performing in-vivo imaging of beating cardiomyocytes in engineered heart tissues and neuronal activity in c. elegans and zebrafish brains.
ABSTRACT
The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.
Subject(s)
Angiotensin-Converting Enzyme 2 , Rhodopsin , Mice , Animals , Action Potentials/physiology , Rhodopsin/genetics , Neurons/physiology , Mutation/geneticsABSTRACT
During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.
Subject(s)
Neurons , Zebrafish , Animals , Mice , Cell Lineage/physiology , Neurons/physiology , Neuroglia , Cell Differentiation/genetics , Neurogenesis/genetics , Gene Expression Regulation, DevelopmentalABSTRACT
The synthesis of a CF3 -rich perfluoropolyether (PFPE) is achieved via the fluoride-catalyzed reaction of hexafluoropropylene oxide (HFPO) with (trifluoromethyl)trimethylsilane (TMSCF3 , so-called Ruppert-Prakash reagent). Nucleophilic addition of a CF3 anion to HFPO affords an acyl fluoride via the ring-opening of HFPO, followed by fluoride elimination. Further addition of CF3 anions to the acyl fluoride gives tertiary perfluoroalkoxide, which attacks HFPO to regenerate an acyl fluoride. Repetition of the sequence via substitution-polymerization affords a new PFPE as a solid, whose structure was confirmed using 19 F NMR spectroscopy, GC-MS, and MALDI-TOF MS analysis. Thermal and X-ray diffraction analyses revealed a crystalline character. To the best of our knowledge, this is the first example of crystalline PFPE. Based on contact-angle measurements, the critical surface tension of this solid PFPE (13.4 mN m-1 ) suggests a water- and oil-repellency of this CF3 -rich PFPE that is higher than that of polytetrafluoroethylene (PTFE; 18.5 mN m-1 ).
Subject(s)
Fluorides , Oxides , Anions , Ethers , Fluorocarbons , Hydrocarbons, Fluorinated/chemistry , Trimethylsilyl CompoundsABSTRACT
Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1 µM to 10 mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed â¼3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.
Subject(s)
Genetic Engineering , Glucose/metabolism , Models, Biological , Animals , Biological Transport , Central Nervous System/metabolism , Drosophila/metabolism , HEK293 Cells , Humans , Imaging, Three-Dimensional , Larva/metabolism , Muscles/metabolism , Neuroglia/metabolism , Proteins/metabolism , Rats, Sprague-Dawley , Zebrafish/metabolismABSTRACT
Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. Here, we present a general approach to engineer electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with positive-going fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further apply this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Action Potentials/physiology , Animals , Luminescent Proteins/metabolism , Neurons/metabolism , Neurosciences/methods , Rhodopsin/chemistry , Rhodopsin/metabolismABSTRACT
We present CLADES (cell lineage access driven by an edition sequence), a technology for cell lineage studies based on CRISPR-Cas9 techniques. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a predetermined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, thereby coupling birth order to reporter expression. This system, which can also be temporally induced by heat shock, enables the temporal resolution of lineage development and can therefore be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, predominantly marking each generation with the corresponding combination of reporters. CLADES therefore offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.
Subject(s)
Cell Lineage/genetics , Animals , Clustered Regularly Interspaced Short Palindromic Repeats , DNA Repair , Drosophila melanogaster , Gene Knock-In Techniques , Genes, Reporter/genetics , Heat-Shock Proteins/genetics , Induced Pluripotent Stem Cells , RNA Editing , Transcriptional Activation , ZebrafishABSTRACT
Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and in vivo, and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging.
Subject(s)
Drug Design , Photosensitizing Agents/chemistry , 3,3'-Diaminobenzidine/chemistry , Animals , Animals, Genetically Modified/metabolism , Cell Line, Tumor , Humans , Larva/metabolism , Ligands , Light , Microphthalmia-Associated Transcription Factor/genetics , Microphthalmia-Associated Transcription Factor/metabolism , Microscopy, Electron , Neurons/chemistry , Neurons/metabolism , Photosensitizing Agents/metabolism , Quantum Theory , Rhodamines/chemistry , Singlet Oxygen/chemistry , Singlet Oxygen/metabolism , Zebrafish/growth & development , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
PURPOSE: Ultrasound (US) is a valuable tool for the evaluation of chronic lateral instability of the ankle; however, the feasibility of US for calcaneofibular ligament (CFL) assessment remains unknown. This study aimed to depict and compare CFL on US in various ankle positions to determine the optimal method for evaluating CFL with US and to interpret US findings using cadaveric specimens. METHODS: The US study included 43 ankles of 25 healthy individuals. The CFL was scanned with US in 20° plantar flexion, neutral position, 20° dorsiflexion and maximum dorsiflexion. The distances between fibula and CFL were compared. The cadaveric study included macroscopic qualitative observation of the dynamic change of CFL in 7 ankles and quantitative observation of the directions of CFL and footprints in 17 ankles. RESULTS: In the US study, the mean distance (mm) between fibula and CFL was 7.3 ± 1.3 in 20° plantar flexion, 6.7 ± 1.6 in neutral position, 4.3 ± 2.5 in 20° dorsiflexion and 3.1 ± 2.1 in maximum dorsiflexion. The more dorsiflexed the ankle was, the shorter the distance between fibula and CFL was (Jonckheere's trend test p < 0.001). In the cadaveric study, the CFL fibres were aligned parallel between the mid-substance and the fibular attachment in maximum dorsiflexion, whilst CFL was reflected and rotated in plantar flexion. CONCLUSIONS: The whole length of the CFL, including its fibular attachment, is more likely to be visualized with US in dorsiflexion than in plantar flexion due to the direction of the CFL at the fibular attachment, which is parallel with the mid-substance in maximum dorsiflexion. LEVEL OF EVIDENCE: IV.
Subject(s)
Lateral Ligament, Ankle/diagnostic imaging , Ultrasonography/methods , Adolescent , Adult , Aged, 80 and over , Ankle , Ankle Joint/diagnostic imaging , Cadaver , Child , Female , Fibula , Healthy Volunteers , Humans , Male , Young AdultABSTRACT
In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.
ABSTRACT
Animal survival requires a functioning nervous system to develop during embryogenesis. Newborn neurons must assemble into circuits producing activity patterns capable of instructing behaviors. Elucidating how this process is coordinated requires new methods that follow maturation and activity of all cells across a developing circuit. We present an imaging method for comprehensively tracking neuron lineages, movements, molecular identities, and activity in the entire developing zebrafish spinal cord, from neurogenesis until the emergence of patterned activity instructing the earliest spontaneous motor behavior. We found that motoneurons are active first and form local patterned ensembles with neighboring neurons. These ensembles merge, synchronize globally after reaching a threshold size, and finally recruit commissural interneurons to orchestrate the left-right alternating patterns important for locomotion in vertebrates. Individual neurons undergo functional maturation stereotypically based on their birth time and anatomical origin. Our study provides a general strategy for reconstructing how functioning circuits emerge during embryogenesis. VIDEO ABSTRACT.
ABSTRACT
Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.
Subject(s)
CRISPR-Cas Systems , DNA Repair , Gene Targeting/methods , Animals , Drosophila , Genetic Techniques , RNA, Guide, Kinetoplastida , Recombinases/genetics , ZebrafishABSTRACT
Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.
Subject(s)
Monitoring, Physiologic/methods , Neuroimaging/methods , Neurons/physiology , Voltage-Sensitive Dye Imaging/methods , Animals , Behavior, Animal , Fluorescence , Fluorescence Resonance Energy Transfer , Genetic Engineering , Larva , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Mesencephalon/cytology , Mesencephalon/physiology , Mice , Optogenetics , Protein Domains , Rhodopsins, Microbial/chemistry , Rhodopsins, Microbial/genetics , Swimming , ZebrafishABSTRACT
Methacrylic esters, represented by methyl methacrylate (MMA), are widely used as commodity chemicals. Here, the one-pot synthesis of methacrylic esters from acetone, a haloform and alcohols in the presence of an organic base is described. Using DBU as the organic base for the reaction of acetone, chloroform and methanol in acetonitrile afforded MMA in 66 % yield. When the solvent was replaced by benzonitrile, the product MMA was successfully purified by distillation. Applicability of this process to various alcohols was also investigated to show ethyl, phenyl, CF3 CH2 , and n-C6 F13 CH2 CH2 esters were obtained in moderate yields. The use of bromoform instead of chloroform resulted in the improvement of the yield, for example, methyl and n-C6 F13 CH2 CH2 esters up to 81 and 70 %, respectively. The reaction with deuterated starting materials acetone-d6 and MeOH-d4 , with DBU in acetonitrile afforded deuterated MMA (MMA-d8 ) in 70 % yield.
ABSTRACT
Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.
Subject(s)
Brain/diagnostic imaging , Neuroimaging , Animals , Brain/anatomy & histology , Dendrites/chemistry , Dendritic Spines/chemistry , Imaging, Three-Dimensional , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , ZebrafishABSTRACT
The emergence of new and increasingly sophisticated behaviors after birth is accompanied by dramatic increase of newly established synaptic connections in the nervous system. Little is known, however, of how nascent connections are organized to support such new behaviors alongside existing ones. To understand this, in the larval zebrafish we examined the development of spinal pathways from hindbrain V2a neurons and the role of these pathways in the development of locomotion. We found that new projections are continually layered laterally to existing neuropil, and give rise to distinct pathways that function in parallel to existing pathways. Across these chronologically layered pathways, the connectivity patterns and biophysical properties vary systematically to support a behavioral repertoire with a wide range of kinematics and dynamics. Such layering of new parallel circuits equipped with systematically changing properties may be central to the postnatal diversification and increasing sophistication of an animal's behavioral repertoire.
Subject(s)
Efferent Pathways/growth & development , Efferent Pathways/physiology , Locomotion , Nerve Net/growth & development , Nerve Net/physiology , Zebrafish/growth & development , Animals , Rhombencephalon/growth & development , Rhombencephalon/physiology , Spine/growth & development , Spine/physiology , Time FactorsABSTRACT
Whole-brain imaging allows for comprehensive functional mapping of distributed neural pathways, but neuronal perturbation experiments are usually limited to targeting predefined regions or genetically identifiable cell types. To complement whole-brain measures of activity with brain-wide manipulations for testing causal interactions, we introduce a system that uses measured activity patterns to guide optical perturbations of any subset of neurons in the same fictively behaving larval zebrafish. First, a light-sheet microscope collects whole-brain data that are rapidly analyzed by a distributed computing system to generate functional brain maps. On the basis of these maps, the experimenter can then optically ablate neurons and image activity changes across the brain. We applied this method to characterize contributions of behaviorally tuned populations to the optomotor response. We extended the system to optogenetically stimulate arbitrary subsets of neurons during whole-brain imaging. These open-source methods enable delineating the contributions of neurons to brain-wide circuit dynamics and behavior in individual animals.
Subject(s)
Behavior, Animal/physiology , Brain Mapping/methods , Brain/physiology , Larva/physiology , Neurons/physiology , Online Systems , Zebrafish/physiology , Animals , Brain/cytology , Neural Pathways , Neurons/cytology , SwimmingABSTRACT
Understanding how neural circuits control behavior requires monitoring a large population of neurons with high spatial resolution and volume rate. Here we report an axicon-based Bessel beam module with continuously adjustable depth of focus (CADoF), that turns frame rate into volume rate by extending the excitation focus in the axial direction while maintaining high lateral resolutions. Cost-effective and compact, this CADoF Bessel module can be easily integrated into existing two-photon fluorescence microscopes. Simply translating one of the relay lenses along its optical axis enabled continuous adjustment of the axial length of the Bessel focus. We used this module to simultaneously monitor activity of spinal projection neurons extending over 60 µm depth in larval zebrafish at 50 Hz volume rate with adjustable axial extent of the imaged volume.