Rapid Mounting of Zebrafish Larvae for Brain Imaging.
Ontology highlight
ABSTRACT:
SUBMITTER: Geng Y
PROVIDER: S-EPMC8716516 | biostudies-literature | 2021 Dec
REPOSITORIES: biostudies-literature
Publications
Rapid Mounting of Zebrafish Larvae for Brain Imaging.
Geng Yijie Y Peterson Randall T RT
Zebrafish 20211201 6
Brain imaging requires mounting of zebrafish larvae in a vertical position, but anesthetized or fixed larvae tend to fall on their sides without external support. Current solution is to manually hold individual larva until liquid agarose solidifies, which is time consuming, labor intensive, and unfriendly to beginners. We developed a method to form larva-shaped slots in agarose gel using a computer numerical controlled manufactured mold. Each slot nearly perfectly fits a larva in its upright pos ...[more]
Similar Datasets
Project description:The zebrafish has emerged as a useful model system for a variety of studies, including the investigation of inflammation and immunity. However, current zebrafish imaging techniques, such as agraose mounting, can be time-consuming and detrimental for long-term imaging. Alternatively, automated sorting and imaging systems can be costly and/or complicated to assemble. Here we describe the Zebrafish Entrapment by Restriction Array (ZEBRA) device, a microfluidic device that can be used to quickly and repeatably position zebrafish embryos in a predictable array using only a pipette. This technique is well suited for use with automated microscope stages leading to decreased imaging time and increased throughput compared to traditional methods. The addition of access ports above the embryo can be used to administer treatments, and potentially wounding or injections. We demonstrate the effectiveness of this device for a neutrophil migration screening application using larvae 3 days post fertilization (dpf) Tg(mpx:dendra2). Larvae were loaded into ZEBRA devices and treated with a neutrophil attractant (LTB4) or LTB4 with and without a PI3K inhibitor, LY294002. Treatment with LY294002 impaired neutrophil motility into the fin induced by LTB4 treatment. The findings report the development of ZEBRA a device that can be used to screen for small molecules that affect leukocyte motility and inflammation using live zebrafish.
| S-EPMC4446983 | biostudies-literature
Project description:Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.
| S-EPMC5755537 | biostudies-other
Project description:Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality(6). Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia(7-9). Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils(10-14). In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts(10, 15-18). To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses(2, 19-21), simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection(22-24). Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection(5). In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection(3, 25). However, only recently have much larger pathogens such as fungi been used to infect larva(5, 23, 26), and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim(25) zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish(5).
| S-EPMC3468152 | biostudies-literature
Project description:The utility of zebrafish is becoming more frequent due to lower costs and high similarities to humans. Zebrafish larvae are attractive subjects for drug screening and drug metabolism research. However, obtaining good quality zebrafish larvae sections for batch samples at designated planes, angles, and locations for comparison purposes is a challenging task. We report here the optimization of fresh frozen zebrafish larvae sectioning for mass spectrometry imaging. We utilized the gelatin solutions that were created at two different temperatures (50 and 85 °C) as embedding media. Gelatin-50 (gelatin created under 50 °C, solid gel under room temperature) was used to make a larvae-shaped mold and gelatin-85 (gelatin created under 85 °C, liquid under room temperature) was used to embed the larvae. H&E staining of sections shows well-preserved morphology and minimal histological interference. More importantly, the position of the larvae was well controlled resulting in more consistent sectioning of the larvae.
| S-EPMC9608760 | biostudies-literature
Project description:Zebrafish play an important role in biological and biomedical research. Traditional in vivo imaging methods for studying zebrafish larvae primarily require fluorescence labeling. In this work, relying on tissue intrinsic optical absorption contrast, we acquired high resolution label-free 3D images of zebrafish larvae by using photoacoustic microscopy (PAM) in vivo. The spatial resolution reaches several microns, allowing the study of microstructures in various living organs. We demonstrated that our method has the potential to be a powerful non-invasive imaging method for studying various small animal models, including zebrafish larvae, Caenorhabditis elegans, frogs and drosophila larvae.
| S-EPMC3269852 | biostudies-other
Project description:Zebrafish, an established model organism in developmental biology, is also a valuable tool for imaging wound healing in space and time with cellular resolution. However, long-term imaging of wound healing poses technical challenges as wound healing occurs over multiple temporal scales. The traditional strategy of larval encapsulation in agarose successfully limits sample movement but impedes larval development and tissue regrowth and is therefore not amenable to long-term imaging of wound healing. To overcome this challenge, we engineered a functionally compartmentalized device, the zebrafish Wounding and Entrapment Device for Growth and Imaging (zWEDGI), to orient larvae for high-resolution microscopy, including confocal and second harmonic generation (SHG), while allowing unrestrained tail development and regrowth. In this device, larval viability was maintained and tail regrowth was improved over embedding in agarose. The quality of tail fiber SHG images collected from larvae in the device was similar to fixed samples but provided the benefit of time lapse data collection. Furthermore, we show that this device was amenable to long-term (>24 h) confocal microscopy of the caudal fin. Finally, the zWEDGI was designed and fabricated using readily available techniques so that it can be easily modified for diverse experimental imaging protocols.
| S-EPMC5312606 | biostudies-literature
Project description:The internal brain dynamics that link sensation and action are arguably better studied during natural animal behaviors. Here, we report on a novel volume imaging and 3D tracking technique that monitors whole brain neural activity in freely swimming larval zebrafish (Danio rerio). We demonstrated the capability of our system through functional imaging of neural activity during visually evoked and prey capture behaviors in larval zebrafish.
| S-EPMC5644961 | biostudies-literature
Project description:Most vertebrates process visual information using elaborately structured photosensory tissues, including the eyes and pineal. However, there is strong evidence that other tissues can detect and respond to photic stimuli. Many reports suggest that photosensitive elements exist within the brain itself and influence physiology and behavior; however, a long-standing puzzle has been the identity of the neurons and photoreceptor molecules involved. We tested whether light cues influence behavior in zebrafish larvae through deep brain photosensors. We found that larvae lacking eyes and pineal perform a simple light-seeking behavior triggered by loss of illumination ("dark photokinesis"). Neuroanatomical considerations prompted us to test orthopedia (otpa)-deficient fish, which show a profound reduction in dark photokinesis. Using targeted genetic ablations, we narrowed the photosensitive region to neurons in the preoptic area. Neurons in this region express several photoreceptive molecules, but expression of the melanopsin opn4a is selectively lost in otpa mutants, suggesting that opn4a mediates dark photokinesis. Our findings shed light on the identity and function of deep brain photoreceptors and suggest that otpa specifies an ancient population of sensory neurons that mediate behavioral responses to light.
| S-EPMC3494761 | biostudies-literature
Project description:Network science has begun to reveal the fundamental principles by which large-scale brain networks are organized, including geometric constraints, a balance between segregative and integrative features, and functionally flexible brain areas. However, it remains unknown whether whole-brain networks imaged at the cellular level are organized according to similar principles. Here, we analyze whole-brain functional networks reconstructed from calcium imaging data recorded in larval zebrafish. Our analyses reveal that functional connections are distance-dependent and that networks exhibit hierarchical modular structure and hubs that span module boundaries. We go on to show that spontaneous network structure places constraints on stimulus-evoked reconfigurations of connections and that networks are highly consistent across individuals. Our analyses reveal basic organizing principles of whole-brain functional brain networks at the mesoscale. Our overarching methodological framework provides a blueprint for studying correlated activity at the cellular level using a low-dimensional network representation. Our work forms a conceptual bridge between macro- and mesoscale network neuroscience and opens myriad paths for future studies to investigate network structure of nervous systems at the cellular level.
| S-EPMC7055648 | biostudies-literature
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 μ-wells in an agarose cast. Each μ-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 μ-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