Imaging In Zebrafish Research Articles

A large field-of-view, single-cell-resolution two- and three-photon microscope for deep and wide imaging

In vivo imaging of large-scale neuronal activity plays a pivotal role in unraveling the function of the brain's circuitry. Multiphoton microscopy, a powerful tool for deep-tissue imaging, has received sustained interest in advancing its speed, field of view and imaging depth. However, to avoid thermal damage in scattering biological tissue, field of view decreases exponentially as imaging depth increases. We present a suite of innovations to optimize three-photon microscopy for large field-of-view imaging at depths unreachable by two-photon microscopy. These techniques enable us to image neuronal activities of transgenic animals expressing protein calcium sensors in a ~ 3.5-mm diameter field-of-view with single-cell resolution in the deepest cortical layer of mouse brains. We further demonstrate simultaneous large field-of-view two-photon and three-photon imaging, subcortical imaging in the mouse brain, and whole-brain imaging in adult zebrafish. The demonstrated techniques can be integrated into typical multiphoton microscopes to enlarge field of view for system-level neural circuit research.

Glial cell derived pathway directs regenerating optic nerve axons toward the CNS midline.

Several RGC intrinsic signaling pathways have been shown to enhance RGC survival and RGC axonal growth after optic nerve injury. Yet an unresolved challenge for regenerating RGC axons is to properly navigate the optic chiasm located at the Central Nervous System midline. Here, we use live-cell imaging in larval zebrafish to show that regrowing RGC axons initiate growth toward the midline and extend along a trajectory similar to their original projection. From a candidate genetic screen, we identify the glycosyltransferase Lh3 to be required during the process of regeneration to direct regrowing RGC axons toward the midline. Moreover, we find that mutants in collagen 18a1 (col18a1), a putative Lh3 substrate, display RGC axonal misguidance phenotypes similar to those we observe in lh3 mutants, suggesting that lh3 may act through col18a1 during regeneration. Finally, we show that transgenic lh3 expression in sox10+ presumptive olig2+ oligodendrocytes located near the optic chiasm restores directed axonal growth. Combined these data identify lh3 and col18a1 as part of a glial derived molecular pathway critical for guiding in vivo regenerating RGC axons towards and across the optic chiasm.

The optimization of sample preparation on zebrafish larvae in vibrational spectroscopy imaging

The zebrafish (Danio rerio) larvae are widely used in biomedical, pharmaceutical, and ecotoxicological studies. Their transparency and translational potential make them particularly valuable for fluorescence imaging. In addition to fluorescence imaging, microspectroscopy, which combines vibrational spectroscopy: Raman or Fourier transform infrared (FT-IR) with microscopy, allows the collection of spatially resolved, label-free information. According to available literature, it was the first application of FT-IR imaging in zebrafish larvae.This study aims to compare different fixation methods for 10-day post-fertilization (dpf) zebrafish larvae using vibrational spectroscopy imaging. Paraformaldehyde (PFA), glutaraldehyde (GA), low temperature, and embedding in gelatin and agarose were investigated. Amides, lipids, and phosphates distribution were more informative in embedded samples but with challenging handling of the sample due to stiffness at −20 °C. FT-IR and Raman mapping revealed that frozen samples had better-preserved tissue structure than chemical fixation. PFA showed uniform amide distribution, while GA treatment exhibited tissue disruptions and denser protein networks in both. Handling of embedded samples is challenging for an operator, but provides more reliable results in developmental biology or disease modeling, compared to chemical treatment.

Small molecule probes with strong one and two-photon excited fluorescence for bioimaging

Two-photon excited fluorescence (TPEF) is widely sought after in bioimaging applications due to its exceptional deep tissue penetration and excellent high spatiotemporal resolution. In this paper, benzothiazole and benzimidazole are linked to N, N-diphenylaniline, respectively, through pyridine vinyl. Two new D-π-A (electron donor-π conjugated bridges-electron acceptor) probes (P1, P2) were designed and synthesized. The molecular structures were completely characterized through FT-IR, 1H NMR, 13C NMR, ESI-MS, and X-ray single crystal diffraction. The probes have good one-photon excited fluorescence and two-photon excited fluorescence. Experimental test results indicate that they can be fluorescence probes for biological imaging of HePG2 and juvenile zebrafish. In addition, the fluorescent probes also have the ability to target and locate cancer cell mitochondria.

The pericardium forms as a distinct structure during heart formation.

The heart integrates diverse cell lineages into a functional unit, including the pericardium, a mesothelial sac that supports heart movement, homeostasis, and immune responses. However, despite its critical roles, the developmental origins of the pericardium remain uncertain due to disparate models. Here, using live imaging, lineage tracking, and single-cell transcriptomics in zebrafish, we find the pericardium forms within the lateral plate mesoderm from dedicated anterior mesothelial progenitors and distinct from the classic heart field. Imaging of transgenic reporters in zebrafish documents lateral plate mesoderm cells that emerge lateral of the classic heart field and among a continuous mesothelial progenitor field. Single-cell transcriptomics and trajectories of hand2-expressing lateral plate mesoderm reveal distinct populations of mesothelial and cardiac precursors, including pericardial precursors that are distinct from the cardiomyocyte lineage. The mesothelial gene expression signature is conserved in mammals and carries over to post-natal development. Light sheet-based live-imaging and machine learning-supported cell tracking documents that during heart tube formation, pericardial precursors that reside at the anterior edge of the heart field migrate anteriorly and medially before fusing, enclosing the embryonic heart to form a single pericardial cavity. Pericardium formation proceeds even upon genetic disruption of heart tube formation, uncoupling the two structures. Canonical Wnt/β-catenin signaling modulates pericardial cell number, resulting in a stretched pericardial epithelium with reduced cell number upon canonical Wnt inhibition. We connect the pathological expression of secreted Wnt antagonists of the SFRP family found in pediatric dilated cardiomyopathy to increased pericardial stiffness: sFRP1 in the presence of increased catecholamines causes cardiomyocyte stiffness in neonatal rats as measured by atomic force microscopy. Altogether, our data integrate pericardium formation as an independent process into heart morphogenesis and connect disrupted pericardial tissue properties such as pericardial stiffness to pediatric cardiomyopathies.

Low-cost Polyethylene Terephthalate Lamination Microfluidics Designs for Multiplexed Zebrafish Imaging.

Zebrafish embryos are transparent and thus uniquely suited for noninvasive intravital imaging of fundamental processes, such as wound healing and immune cell migration. Microfluidic devices are used for entrapment to support long-term imaging of multicellular organisms, including zebrafish. However, the fabrication of these devices using soft lithography requires specialized facilities and competency in 3D printing, which may not be accessible to every lab. Our adaptation of a previously developed low-cost polyethylene terephthalate lamination method for constructing microfluidic devices increases accessibility by enabling design fabrication and iteration for a fraction of the technical investment of conventional techniques. We use a device made with this method, the Rotational Assistant for Danio Imaging of Subsequent Healing (RADISH), to accommodate drug treatment, manual wounding, and long-term imaging of up to four embryos in the same field of view. With this new design, we successfully capture gross morphological characteristics of the calciumsignal around laser ablation and manual transection wounds for multiple embryos in the 2 h immediately following injury, as well as neutrophil recruitment to the wound edge for 24 h.

A novel high signal-to-noise ratio fluorescent probe for real-time mitochondrial viscosity detection and imaging in vitro and in vivo.

Mitochondrial viscosity serves as a critical indicator for assessing mitochondrial functionality and offers valuable insights into cellular homeostasis. Continuous, real-time monitoring of mitochondrial viscosity is indispensable for understanding and diagnosing diseases associated with these dynamic changes. In this study, we introduce a novel mitochondrial viscosity-responsive probe named "JL-JC" which is designed by using a molecular strategy, with a classic "D-π-A" molecular structure. Leveraging the distinctive twisted intramolecular charge transfer (TICT) properties of the probe, JL-JC exhibits exceptional sensitivity and a high signal-to-noise ratio, enabling precise detection of viscosity variations within its microenvironment while remaining unaffected by other factors. Upon rapid cellular uptake, JL-JC can efficiently evaluate the mitochondrial viscosity changes under diverse physiological and pathological conditions. Notably, this probe also enables viscosity imaging in zebrafish, offering insights into mitochondrial states in vivo. Our findings present JL-JC as a promising tool and potential diagnostic platform for mitochondria-related diseases.

Decreased GABA levels during development result in increased connectivity in the larval zebrafish tectum.

γ-aminobutyric acid (GABA) is an abundant neurotransmitter that plays multiple roles in the vertebrate central nervous system (CNS). In the early developing CNS, GABAergic signaling acts to depolarize cells. It mediates several aspects of neural development, including cell proliferation, neuronal migration, neurite growth, and synapse formation, as well as the development of critical periods. Later in CNS development, GABAergic signaling acts in an inhibitory manner when it becomes the predominant inhibitory neurotransmitter in the brain. This behavior switch occurs due to changes in chloride/cation transporter expression. Abnormalities of GABAergic signaling appear to underlie several human neurological conditions, including seizure disorders. However, the impact of reduced GABAergic signaling on brain development has been challenging to study in mammals. Here we take advantage of zebrafish and light sheet imaging to assess the impact of reduced GABAergic signaling on the functional circuitry in the larval zebrafish optic tectum. Zebrafish have three gad genes: two gad1 paralogs known as gad1a and gad1b, and gad2. The gad1b and gad2 genes are expressed in the developing optic tectum. Null mutations in gad1b significantly reduce GABA levels in the brain and increase electrophysiological activity in the optic tectum. Fast light sheet imaging of genetically encoded calcium indicator (GCaMP)-expressing gab1b null larval zebrafish revealed patterns of neural activity that were different than either gad1b-normal larvae or gad1b-normal larvae acutely exposed to pentylenetetrazole (PTZ). These results demonstrate that reduced GABAergic signaling during development increases functional connectivity and concomitantly hyper-synchronization of neuronal networks.

Live imaging in zebrafish reveals tissue-specific strategies for amoeboid migration.

Amoeboid cells like leukocytes can enter and migrate within virtually every tissue of the body, even though tissues vary widely in their chemical and mechanical composition. Here, we imaged motile T cells as they colonized peripheral tissues during zebrafish development to ask if cells tailor their migration strategy to their local tissue environment. We found that T cells in most sites migrated with f-actin-rich leading-edge pseudopods, matching how they migrate in vitro . T cells notably deviated from this strategy in the epidermis, where they instead migrated using a rearward concentration of f-actin and stable leading-edge blebs. This mode of migration occurs under planar confinement in vitro , and we correspondingly found the stratified keratinocyte layers of the epidermis impose planar-like confinement on leukocytes in vivo . By imaging the same cell type across the body, our data collectively indicates that cells adapt their migration strategy to navigate different tissue geometries in vivo .

Construction of a highly specific fluorescence “turn-on” probe for H2S detection and imaging in drug-induced live cells, zebrafish and mice arthritis models

Quantitatively and selectively detecting the biomarker of hydrogen sulfide (H2S) in arthritis diseases is of great significance for the early diagnosis and treatment of arthritis. Modern medical studies show that H2S as a biomarker is involved in the development of inflammation. In this work, a new highly specific fluorescence “turn-on” probe JMD-H2S was tailored for H2S detection and imaging in drug-induced live cells, zebrafish and mice arthritis models, which utilized pyrazoline molecule as the fluorescence signal reporter group and 2,4-dinitrophenyl ether group (DNB) with strong intramolecular charge transfer (ICT) effect as the H2S recognition moiety and fluorescence quenching group. JMD-H2S showed a fast response time (<60 s), a large fluorescence response ratio (enhanced ∼20 folds) at I453/I0, excellent sensitivity toward H2S over other analytes, and an outstanding limit of detection (LOD) as low as 25.3 nM. In addition, JMD-H2S has been successfully applied for detecting and imaging H2S in drug-induced live cells, zebrafish, and mice arthritis models with satisfactory results, suggesting it can be used as a robust molecular tool for investigating the occurrence and development of H2S and arthritis.

Super-resolution imaging lysosome vesicles and establishing a gallbladder-visualizable zebrafish model via a fluorescence probe

Advanced probes for imaging viscous lipids microenvironment in vitro and in vivo are desirable for the study of membranous organelles and lipids traffic. Herein, a reaction-based dihydroquinoline probe (DCQ) was prepared via linking a diethylamino coumarin fluorophore with a N-methylquinoline moiety. DCQ is stable in low viscous aqueous mediums and exhibits green fluorescence, which undergoes fast autoxidation in high viscous mediums to form a fluorescent product with deep-red to near-infrared (NIR) emission, rendering the ability for dual-color imaging. Living cell imaging indicated that DCQ can effectively stain lysosomal membranes with deep-red fluorescence. Super-resolution imaging of lysosome vesicles has been achieved by DCQ and stimulated emission depletion (STED) microscopy. In addition, DCQ realizes multiple organs imaging in zebrafish, whose dual-color emission can perfectly discriminate zebrafish's yolk sac, digestive tract and gallbladder. Most importantly, DCQ has been successfully used to establish a gallbladder-visualizable zebrafish model for the evaluation of drug stress.

Advancing Alzheimer's Research With Zebrafish Models: Current Insights, Addressing Challenges, and Charting Future Courses.

Alzheimer's disease (AD) is a neurological condition that progressively impairs cognitive function and results in memory loss.Despite substantial research efforts, little is known about the specific processes driving AD, and there are few proven therapies. Because of their physiological and genetic resemblance to humans, zebrafish (Danio rerio) have become an important model organism for furthering research on AD.This abstract discusses the difficulties faced, looks at the insights currently garnered from zebrafish models, and suggests future research options. AD knowledge has greatly benefited from the use of zebrafish models. Transgenic zebrafish that express human AD-associated genes, such as tau and amyloid precursor protein (APP), display tau neurofibrillary tangles (NFTs) and amyloid-beta (Aβ) plaques, two of the disease's main clinical characteristics. These models have clarified the roles of oxidative stress, inflammation, and calcium homeostasis in the course of AD and allowed for the purpose of high-throughput screening of potential therapeutic agents.Understanding the growth and deterioration of neurons has been greatly aided by real-time zebrafish imaging. Fully using zebrafish models in AD research requires addressing a number of issues. The dissimilarities in zebrafish anatomy and physiology from humans, the difficulty of developing models that replicate progressive and late-onset AD (LOAD), and the requirement for standardized procedures to evaluate alterations in zebrafish cognition and behavior are a few issues.Furthermore, variations in the genetic makeup of zebrafish strains might affect the results of experiments. Future directions include developing standardized behavioral assays and cognitive tests, working together to create extensive databases of zebrafish genetic and phenotypic data, and using genetic engineering techniques like CRISPR/Cas9 to create more complex zebrafish models. Combining zebrafish models with other model species helps expedite the conversion of research results into therapeutic applications and offers a more thorough knowledge of AD.To sum up, zebrafish models have made a substantial contribution to Alzheimer's research by offering insightful information on the causes of the illness and possible therapies. By tackling present issues and formulating a planned future path, we can improve the use of zebrafish to decipher the mysteries of Alzheimer's and help create successful treatments.

Bodipy-Based highly sensitive hydrogen sulfide fluorescent probe and its fluorescence imaging in cells and zebrafish

Hydrogen sulfide (H2S) is a crucial endogenous gasotransmitter that plays a role in various physiological and pathological processes. Therefore, accurate and rapid monitoring of H2S in organisms is highly significant for understanding the underlying pathological mechanisms and facilitating early diagnosis of related diseases. In this study, we developed a novel fluorescent probe, B-CHO-NO2, based on a bodipy fluorophore, which exhibits excellent sensitivity and selectivity towards H2S. The design of the probe exploits the nucleophilicity of H2S by introducing a formyl group as the ortho-participating moiety, significantly enhancing the reaction rate with H2S. In cellular and zebrafish models, the probe B-CHO-NO2 successfully achieved fluorescence imaging of endogenous and exogenous H2S. The development of probe B-CHO-NO2 provides a powerful tool for biological studies of H2S and diagnosis of related diseases.

“Double-locked” fluorescent probe based on a push-pull structure for detecting HClO/H2O2

Fluorophores with “push-pull” electronic structures can quench their fluorescence by shielding either part, and can be easily used to construct “double-locked” fluorescent probes. At present, research in this area is still rare. To demonstrate its feasibility, here we selected 6-acetyl-2-naphthol, a simple fluorophore with a characteristic “push-pull” structure (hydroxy-ketone) to construct a double-locked probe for the detection of HClO/H2O2. Specifically, 1,3-oxathiolane was employed as an HClO-responsive group to shield the ketone (pull group), while the hydroxy (push group) was protected by an H2O2-responsive boronate ester group, resulting in the HClO/H2O2-specific probe Sa1. In vitro experiments confirmed that Sa1 emits fluorescence only in the presence of both HClO and H2O2, exhibiting remarkable concentration- and time-dependent fluorescence responses. Additionally, Sa1 displayed excellent selectivity and low toxicity. Finally, through cell and zebrafish imaging experiments, the dual detection capability of Sa1 in biological systems was validated.

Activatable Fluorescent Probe for Studying Drug-Induced Senescence In Vitro and In Vivo.

Aging represents a significant risk factor for compromised tissue function and the development of chronic diseases in the human body. This process is intricately linked to oxidative stress, with HClO serving as a vital reactive oxygen species (ROS) within biological systems due to its strong oxidative properties. Hence, conducting a thorough examination of HClO in the context of aging is crucial for advancing the field of aging biology. In this work, we successfully developed a fluorescent probe, OPD, tailored specifically for detecting HClO in senescent cells and in vivo. Impressively, OPD exhibited a robust reaction with HClO, showcasing outstanding selectivity, sensitivity, and photostability. Notably, OPD effectively identified HClO in senescent cells for the first time, confirming that DOX- and ROS-induced senescent cells exhibited higher HClO levels compared to uninduced normal cells. Additionally, in vivo imaging of zebrafish demonstrated that d-galactose- and ROS-stimulated senescent zebrafish displayed elevated HClO levels compared to normal zebrafish. Furthermore, when applied to mouse tissues and organs, OPD revealed increased fluorescence in the organs of senescent mice compared to their nonsenescent counterparts. Our findings also illustrated the probe's potential for detecting changes in HClO content pre- and post-aging in living mice. Overall, this probe holds immense promise as a valuable tool for in vivo detection of HClO and for studying aging biology in live organisms.

Dicyanisophorone-based viscosity fluorescent probe and its fluorescence imaging in cells and zebrafish

Cellular viscosity is a critical parameter for maintaining normal cellular functions, and its abnormalities are associated with the occurrence and development of various diseases. Therefore, real-time monitoring of cellular viscosity is of significant importance for understanding viscosity-related biological functions and revealing the pathological mechanisms of diseases. In this study, we developed a novel fluorescence probe (IP-HBO) by hybridizing dicyanisophorone with 2-(2-hydroxyphenyl)benzoxazole for monitoring of cellular microenvironment and viscosity changes in zebrafish. The probe IP-HBO combines the principles of molecular rotors and enhanced solid-state fluorescence properties, demonstrating highly specific responsiveness to viscosity, long-wavelength emission, large Stokes shift, and excellent photostability. It has been successfully employed for fluorescence imaging of viscosity in live HeLa cells and zebrafish. The construction of this probe provides a new tool for gaining insights into viscosity-related biological processes and disease mechanisms within cells.

Long-working-distance high-collection-efficiency three-photon microscopy for in vivo long-term imaging of zebrafish and organoids

Zebrafish and organoids, crucial for complex biological studies, necessitate an imaging system with deep tissue penetration, sample protection from environmental interference, and ample operational space. Traditional three-photon microscopy is constrained by short-working-distance objectives and falls short. Our long-working-distance high-collection-efficiency three-photon microscopy (LH-3PM) addresses these challenges, achieving a 58% fluorescence collection efficiency at a 20mm working distance. LH-3PM significantly outperforms existing three-photon systems equipped with the same long working distance objective, enhancing fluorescence collection and dramatically reducing phototoxicity and photobleaching. These improvements facilitate accurate capture of neuronal activity and an enhanced detection of activity spikes, which are vital for comprehensive, long-term imaging. LH-3PM's imaging of epileptic zebrafish not only showed sustained neuron activity over an hour but also highlighted increased neural synchronization and spike numbers, marking a notable shift in neural coding mechanisms. This breakthrough paves the way for new explorations of biological phenomena in small model organisms.

Satellite glial cell manipulation prior to axotomy enhances developing dorsal root ganglion central branch regrowth into the spinal cord.

The central and peripheral nervous systems (CNS and PNS, respectively) exhibit remarkable diversity in the capacity to regenerate following neuronal injury with PNS injuries being much more likely to regenerate than those that occur in the CNS. Glial responses to damage greatly influence the likelihood of regeneration by either promoting or inhibiting axonal regrowth over time. However, despite our understanding of how some glial lineages participate in nerve degeneration and regeneration, less is known about the contributions of peripheral satellite glial cells (SGC) to regeneration failure following central axon branch injury of dorsal root ganglia (DRG) sensory neurons. Here, using in vivo, time-lapse imaging in larval zebrafish coupled with laser axotomy, we investigate the role of SGCs in axonal regeneration. In our studies we show that SGCs respond to injury by relocating their nuclei to the injury site during the same period that DRG neurons produce new central branch neurites. Laser ablation of SGCs prior to axon injury results in more neurite growth attempts and ultimately a higher rate of successful central axon regrowth, implicating SGCs as inhibitors of regeneration. We also demonstrate that this SGC response is mediated in part by ErbB signaling, as chemical inhibition of this receptor results in reduced SGC motility and enhanced central axon regrowth. These findings provide new insights into SGC-neuron interactions under injury conditions and how these interactions influence nervous system repair.

EVIDENCE FOR AUDITORY STIMULUS-SPECIFIC ADAPTATION BUT NOT DEVIANCE DETECTION IN LARVAL ZEBRAFISH BRAINS.

Animals receive a constant stream of sensory input, and detecting changes in this sensory landscape is critical to their survival. One signature of change detection in humans is the auditory mismatch negativity (MMN), a neural response to unexpected stimuli that deviate from a predictable sequence. This process requires the auditory system to adapt to specific repeated stimuli while remaining sensitive to novel input (stimulus-specific adaptation). MMN was originally described in humans, and equivalent responses have been found in other mammals and birds, but it is not known to what extent this deviance detection circuitry is evolutionarily conserved. Here we present the first evidence for stimulus-specific adaptation in the brain of a teleost fish, using whole-brain calcium imaging of larval zebrafish at single-neuron resolution with selective plane illumination microscopy. We found frequency-specific responses across the brain with variable response amplitudes for frequencies of the same volume, and created a loudness curve to model this effect. We presented an auditory 'oddball' stimulus in an otherwise predictable train of pure tone stimuli, and did not find a population of neurons with specific responses to deviant tones that were not otherwise explained by stimulus-specific adaptation. Further, we observed no deviance responses to an unexpected omission of a sound in a repetitive sequence of white noise bursts. These findings extend the known scope of auditory adaptation and deviance responses across the evolutionary tree, and lay groundwork for future studies to describe the circuitry underlying auditory adaptation at the level of individual neurons.

A high-stable and fast-responsive lysosome-targeted probe for detection of endogenous and exogenous HClO in living cells and its application in zebrafish imaging

HClO plays a vital role in the process of lysosome degradation of harmful substances within cells, thus it is urgently needed to develop a powerful imaging tool for detecting HClO in lysosomes. Herein, morpholine was introduced into 1,8-naphthoimide fluorophore as a lysosome targeting unit, and the 3-position of naphthalimide was modified with oxime moiety (CNOH) to synthesize a probe Nap-HClO for the detection of lysosomal HClO. The detection of HClO by Nap-HClO had been studied through theoretical calculations, which well verified the sensing mechanism of the probe. A comparison between the calculation results and experimental data revealed a close correlation between the two, indicating that Nap-HClO possesses theoretically predictable properties for HClO detection. In experiments on endogenous and exogenous HClO in cellular lysosomes, as well as exogenous HClO in zebrafish, Nap-HClO successfully monitored HClO. Nap-HClO exhibited high selectivity, rapid response (<1 min), low detection limit (∼6.8 × 10−6 M), and excellent stability in HClO detection, making it an excellent tool for detecting lysosomal HClO in organisms.