A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos.
Ontology highlight
ABSTRACT:
SUBMITTER: Upadhyay S
PROVIDER: S-EPMC8411746 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundDevelopmental biology relies to a large extent on the observation and comparison of phenotypic traits through time using high resolution microscopes. In this context, transparent model organisms such as the zebrafish Danio rerio in which developing tissues and organs can be easily observed and imaged using fluorescent proteins have become very popular. One limiting factor however is the acquisition of a sufficient amount of data, in standardized and reproducible conditions, to allow robust quantitative analysis. One way to improve this is by developing mounting methods to increase the number of embryos that can be imaged simultaneously in near-to-identical orientation.ResultsHere we present an improved mounting method allowing semi-automated and high-content imaging of zebrafish embryos. It is based on a 3D-printed stamp which is used to create a 2D coordinate system of multiple ?-wells in an agarose cast. Each ?-well models a negative of the average zebrafish embryo morphology between 22 and 96?h-post-fertilization. Due to this standardized and reproducible arrangement, it is possible to define a custom well plate in the respective imaging software that allows for a semi-automated imaging process. Furthermore, the improvement in Z-orientation significantly reduces post-processing and improves comparability of volumetric data while reducing light exposure and thus photo-bleaching and photo-toxicity, and improving signal-to-noise ratio (SNR).ConclusionsWe present here a new method that allows to standardize and improve mounting and imaging of embryos. The 3D-printed stamp creates a 2D coordinate system of ?-wells in an agarose cast thus standardizing specimen mounting and allowing high-content imaging of up to 44 live or mounted zebrafish embryos simultaneously in a semi-automated, well-plate like manner on inverted confocal microscopes. In summary, image data quality and acquisition efficiency (amount of data per time) are significantly improved. The latter might also be crucial when using the services of a microscopy facility.
| S-EPMC6805687 | biostudies-literature
Project description:BackgroundThe zebrafish Danio rerio is an important model system for drug discovery and to study cardiovascular development. Using a laser-scanning confocal microscope, we have developed a non-invasive method of measuring cardiac performance in zebrafish embryos and larvae that obtains cardiovascular parameters similar to those obtained using Doppler echocardiography in mammals. A laser scan line placed parallel to the path of blood in the dorsal aorta measures blood cell velocity, from which cardiac output and indices of vascular resistance and contractility are calculated.ResultsThis technique, called laser-scanning velocimetry, was used to quantify the effects of pharmacological, developmental, and genetic modifiers of cardiac function. Laser-scanning velocimetry was applied to analyze the cardiovascular effects of morpholino knockdown of osmosensing scaffold for MEKK3 (OSM), which when mutated causes the human vascular disease cerebral cavernous malformations. OSM-deficient embryos had a constricted aortic arch and markedly increased peak cell velocity, a characteristic indicator of aortic stenosis.ConclusionThese data validate laser-scanning velocimetry as a quantitative tool to measure cardiovascular performance for pharmacological and genetic analysis in zebrafish, which requires no specialized equipment other than a laser-scanning confocal microscope.
| S-EPMC1955438 | biostudies-literature
Project description:Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.
| S-EPMC7241436 | biostudies-literature
Project description:In vivo functional imaging at single-neuron resolution is an important approach to visualize biological processes in neuroscience. Light sheet microscopy (LSM) is a cutting edge in vivo imaging technique that provides micron-scale spatial resolution at high frame rate. Due to the scattering and absorption of tissue, however, conventional LSM is inadequate to resolve cells because of the attenuated signal to noise ratio (SNR). Using dual-beam illumination and confocal dual-slit detection, here a dual-slit confocal LSM is demonstrated to obtain the SNR enhanced images with frame rate twice as high as line confocal LSM method. Through theoretical calculations and experiments, the correlation between the slit's width and SNR was determined to optimize the image quality. In vivo whole brain structural imaging stacks and the functional imaging sequences of single slice were obtained for analysis of calcium activities at single-cell resolution. A two-fold increase in imaging speed of conventional confocal LSM makes it possible to capture the sequence of the neurons' activities and help reveal the potential functional connections in the whole zebrafish's brain.
| S-EPMC4467708 | biostudies-other
Project description:Ice crystals nucleate and grow when a water solution is cooled below its freezing point. The growth velocities and morphologies of the ice crystals depend on many parameters, such as the temperature of ice growth, the melting temperature, and the interactions of solutes with the growing crystals. Three types of morphologies may appear: dendritic, cellular (or fingerlike), or the faceted equilibrium form. Understanding and controlling which type of morphology is formed is essential in several domains, from biology to geophysics and materials science. Obtaining, in situ, three dimensional observations without introducing artifacts due to the experimental technique is nevertheless challenging. Here we show how we can use laser scanning confocal microscopy to follow in real-time the growth of smoothed and faceted ice crystals in zirconium acetate solutions. Both qualitative and quantitative observations can be made. In particular, we can precisely measure the lateral growth velocity of the crystals, a measure otherwise difficult to obtain. Such observations should help us understand the influence of the parameters that control the growth of ice crystals in various systems.
| S-EPMC5131326 | biostudies-literature
Project description:PURPOSE:Retinal microglia have been implicated in the pathogenesis of various retinal diseases, but their basic function and cellular phenotype remain incompletely understood. Here, the authors used a novel ex vivo retinal imaging preparation to examine the behavioral phenotype of living retinal microglia in intact tissue and in response to injury. METHODS:Fluorescence-labeled microglia in retinal explants from CX3CR1(+/GFP) transgenic mice were observed using time-lapse confocal imaging. High spatial and temporal resolution imaging parameters were used to follow dynamic microglial behavior in real time. RESULTS:Under normal conditions, resting retinal microglia are not static in structure but instead exhibit extensive structural dynamism in their cellular processes. Process movements are highly random in direction but are balanced to maintain overall cellular symmetry and arbor size. At rest, however, these exuberant process movements do not result in overt cellular migration. After focal laser injury, microglial processes increase significantly in their motility and direct themselves toward the injury site. Microglia rapidly transition their morphologies from symmetric to polarized toward the laser lesion. Microglia also transition from a fixed to a migratory phenotype, translocating through tissue while retaining their ramified morphology. CONCLUSIONS:Retinal microglia normally occupying uninjured tissue display a continuous, dynamic behavior that suggests functions of tissue surveillance and intercellular communication. Microglial behavior is highly regulated by, and immediately responsive to, focal tissue injury and may constitute a therapeutic cellular response to focal laser photocoagulation. Ex vivo live imaging in the retina is an experimental approach well suited to the study of dynamic aspects of microglial physiology.
| S-EPMC2652634 | biostudies-literature
Project description:Zebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ?RD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivo temporal monitoring of the wound response in living zebrafish embryos. Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis of fully intact and living zebrafish embryos.
| S-EPMC7710741 | biostudies-literature
Project description:Biofilms are spatially organized communities of microorganisms embedded in a self-produced organic matrix, conferring to the population emerging properties such as an increased tolerance to the action of antimicrobials. It was shown that some bacilli were able to swim in the exogenous matrix of pathogenic biofilms and to counterbalance these properties. Swimming bacteria can deliver antimicrobial agents in situ, or potentiate the activity of antimicrobial by creating a transient vascularization network in the matrix. Hence, characterizing swimmer trajectories in the biofilm matrix is of particular interest to understand and optimize this new biocontrol strategy in particular, but also more generally to decipher ecological drivers of population spatial structure in natural biofilms ecosystems. In this study, a new methodology is developed to analyze time-lapse confocal laser scanning images to describe and compare the swimming trajectories of bacilli swimmers populations and their adaptations to the biofilm structure. The method is based on the inference of a kinetic model of swimmer populations including mechanistic interactions with the host biofilm. After validation on synthetic data, the methodology is implemented on images of three different species of motile bacillus species swimming in a Staphylococcus aureus biofilm. The fitted model allows to stratify the swimmer populations by their swimming behavior and provides insights into the mechanisms deployed by the micro-swimmers to adapt their swimming traits to the biofilm matrix.
| S-EPMC9273218 | biostudies-literature
Project description:Melanoma is the deadliest type of skin cancer, characterized by high cellular heterogeneity which contributes to therapy resistance and unpredictable disease outcome. Recently, by correlating Reflectance-Confocal-Microscopy (RCM) morphology with histopathological type, we identified four distinct melanoma-subtypes: dendritic-cell (DC), round-cell (RC), dermal-nest (DN), and combined-type (CT) melanomas. Our results demontsrate that each melanoma subtypes has a distinct biological and gene expression profile, related to tumor aggressiveness, confirming that RCM can be a dependable tool for in vivo detecting different types of melanoma and for early diagnostic screening
2021-11-26 | GSE174395 | GEO
Project description:BACKGROUND: Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. METHODS: Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). RESULTS: We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. CONCLUSION: The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes.
| S-EPMC2515298 | biostudies-literature