Imaging In Zebrafish Research Articles

A novel fluorescent probe for efficient detection of fluoride ions in living animal and plant tissues.

This work presents the design and synthesis of a new fluorescent probe IF-Br-F for the specific detection of fluoride ions. The IF-Br-F probe has excellent fluorescence properties, and the mechanism of the probe response to fluoride ions was successfully verified via HRMS and DFT calculations. IF-Br-F has high sensitivity and low detection limit (5.82 × 10-7 mol L-1) and successfully detects fluoride ions in actual water samples. The probe can be applied to fluorescence imaging of zebrafish, cells, and Arabidopsis roots and exhibits low cytotoxicity and good biocompatibility.

Monoamine oxidase A probe synthesis and fluorescence imaging in cells and zebrafish based on AIE mechanism

Monoamine oxidase A (MAO-A) is a crucial mammalian flavoenzyme that plays a significant role in various neurodegenerative diseases such as depression, Parkinson’s disease, schizophrenia, and epilepsy, as well as cancer, cardiac disorders, and liver disease. Thus, a real-time and targeted MAO-A detection approach is desperately needed to study the onset and management of disorders linked to MAO-A. In this study, we have successfully synthesized a red-emitting fluorescent probe AIE-M, which is based on the specificity of MAOs for the structural catalysis of propylamine group substrates. This probe utilizes quinoline-malononitrile (QM) with AIE properties as the fluorescent master. The probe containing QM exhibits excellent aggregation-induced luminescence (AIE), biocompatibility, and high photostability, enabling specific recognition of MAO-A. Moreover, it can be employed for the detection and imaging of endogenous MAO-A in neuroblastoma cells and zebrafish, thereby offering a novel research instrument for diagnosing associated ailments.

Highly Efficient Red Thermally Activated Delayed Fluorescence Nanoparticles for Real‐Time in Vivo Time‐Resolved Luminescence Imaging

AbstractThermally activated delayed fluorescence (TADF) nanoparticles are used importantly in time‐resolved luminescence imaging for eliminating the background signals from scattering and short‐lived autofluorescence. However, TADF nanoparticles are seldom used for real‐time time‐resolved luminescence imaging, due to their limited luminescence efficiency and lifetimes. To detect delayed fluorescence, multiple excitation cycles with adequate delay times are usually required, leading to long detecting durations and low imaging speed. Herein, highly efficient red TADF nanoparticles are developed through doping a guest molecule, TPAAQ (2,6‐bis[4‐(diphenylamino) phenyl] anthraquinone), in a host (4,4′‐bis(carbazol‐9‐yl)biphenyl, CBP) matrix. With a low doping concentration, the nanoparticles can exhibit obvious TADF with luminescence lifetimes over 0.1 ms and photoluminescence quantum yield up to 35%. A cell‐penetrating peptide is used together with the amphiphilic compound for assembling nanoparticles, which can easily penetrate cells and greatly increase the TADF signals for luminescence lifetime imaging. Thanks to the long‐lived and highly efficient TADF, real‐time in vivo time‐gated luminescence imaging of zebrafish is realized on a chopper‐based wide‐field microscope. This low‐cost time‐resolved luminescence imaging method showed a great potential for real‐time detection of life activities in many organisms with high autofluorescence.

A structural optimized fluorescent probe for monitoring hydrogen sulfide in cells and zebrafish

In this work, we reported a fluorescent probe Fur-SH, a derivative of benzofuranone, which was used to detect H2S in living cells and zebrafish. Based on the three structural characteristics of the probe, the effects of different structural modifications on the optical properties of the fluorophore were compared. Then, the fluorophore Fur-OH was synthesized by modifying diethylamino group with benzofuranone as the main skeleton. With 2,4-dinitrofluorobenzene as the recognition group and diethylamino as the electron donor, the push–pull electron effect occurred with nitro group, which led to fluorescence quenching, and an openable fluorescent probe Fur-SH was formed. The probe Fur-SH (λex = 510 nm; λem = 570 nm) had the advantages of smaller full width at half maxima, rapid response (5 min) and wide pH window. The quantitative properties of the probe were excellent, reaching saturation at 50 equivalents of substrate. The probe Fur-SH showed high sensitivity to H2S, with LOD of 48.9 nM and LOQ of 50 nM. At present, the probe Fur-SH had been applied to fluorescence imaging of MCF-7 cells and zebrafish. By comparing the effects of different structures on the optical properties of fluorophores, this work was expected to be helpful to the development of fluorescent probes in the future.

Synthesis of a super-low detection limit fluorescent probe for Al3+ and its application in fluorescence imaging of zebrafish and cells

A novel fluorescent probe N'-(2-hydroxybenzylidene)-indole-3-formylhydrazine (JHK) was designed and synthesized based on the condensation reaction of indole-3-formylhydrazine and salicylaldehyde. The probe JHK solution could highly selectively recognize Al3+ by the obvious fluorescence enhancement (288-fold) after adding Al3+. And the probe solution with Al3+ had a very high fluorescence quantum yield (89.29 %). The detection limit was calculated to be 1.135 nM, which was significantly lower than many reported detection limits, indicating that the probe JHK had pretty good sensitivity. The ratio of JHK to Al3+ (1:1) and the sensing mechanism were determined by Job's plot, 1H NMR spectra, FTIR spectra, ESI-MS and Gaussian calculation. The probe solution and medium-speed filter paper were successfully used to make test papers for more convenient detection of Al3+. Furthermore, the probe JHK had been successfully applied to the detection of Al3+ in real water, zebrafish and living cells.

Deep learning-based inpainting of saturation artifacts in optical coherence tomography images

Limited by the dynamic range of the detector, saturation artifacts usually occur in optical coherence tomography (OCT) imaging for high scattering media. The available methods are difficult to remove saturation artifacts and restore texture completely in OCT images. We proposed a deep learning-based inpainting method of saturation artifacts in this paper. The generation mechanism of saturation artifacts was analyzed, and experimental and simulated datasets were built based on the mechanism. Enhanced super-resolution generative adversarial networks were trained by the clear–saturated phantom image pairs. The perfect reconstructed results of experimental zebrafish and thyroid OCT images proved its feasibility, strong generalization, and robustness.

A selenium-based fluorescent probe for the sensitive detection of hypochlorous acid and its application in cell and zebrafish imaging

Hypochlorous acid (HOCl) is a significant reactive oxygen species within biological systems, and its anomalous metabolism has been linked to a range of diseases. Therefore, it is crucial to track and monitor the concentration of HOCl in living organisms to understand its biological functions and study its pathogenic mechanisms. In this study, we developed a photoinduced electron transfer (PeT) mechanism-based benzo-fused bodipy fluorescence probe, named BBy-Se, for highly sensitive and selective detection of HOCl in aqueous solutions. BBy-Se demonstrated a good turn-on fluorescence response to HOCl by specifically oxidizing the selenium atom. It exhibited significant Stokes shift (82 nm), low detection limit (10.9 nM) and rapid response (less than 10 s). Furthermore, the probe was successfully employed for real-time fluorescence imaging of HOCl in living HeLa cells and zebrafish, as demonstrated by the bioimaging results. The development of BBy-Se presents a new approach to explore the biological roles of HOCl and offers a valuable tool for studying its function in diseases.

Xanthene-based Hg2+ fluorescent probe for detection of Hg2+ in water/food samples, as well as imaging of live cells, zebrafish and tobacco seedlings

In this paper, an Hg2+ detection probe, HOS, was prepared with a xanthene as the parent fluorophore. Hg2+-initiated thioacetal deprotection reaction is the detection mechanism of this probe. After testing, the probe HOS was able to accurately determine Hg2+ with a detection limit of 36 nM. It was successfully applied to the detection of Hg2+ in different water samples and shrimp samples, meanwhile, the filter paper strips prepared by HOS were obviously changed from light yellow to dark yellow under daylight, and from green to yellow under 365 nm UV light. Furthermore, probe HOS enabled Hg2+ bioimaging experiments on HepG2 cells, zebrafish and tobacco seedlings under laser confocal microscopy.

The Reissner fiber under tension in vivo shows dynamic interaction with ciliated cells contacting the cerebrospinal fluid.

The Reissner fiber (RF) is an acellular thread positioned in the midline of the central canal that aggregates thanks to the beating of numerous cilia from ependymal radial glial cells (ERGs) generating flow in the central canal of the spinal cord. RF together with cerebrospinal fluid (CSF)-contacting neurons (CSF-cNs) form an axial sensory system detecting curvature. How RF, CSF-cNs and the multitude of motile cilia from ERGs interact in vivo appears critical for maintenance of RF and sensory functions of CSF-cNs to keep a straight body axis, but is not well-understood. Using in vivo imaging in larval zebrafish, we show that RF is under tension and resonates dorsoventrally. Focal RF ablations trigger retraction and relaxation of the fiber's cut ends, with larger retraction speeds for rostral ablations. We built a mechanical model that estimates RF stress diffusion coefficient D at 5 mm2/s and reveals that tension builds up rostrally along the fiber. After RF ablation, spontaneous CSF-cN activity decreased and ciliary motility changed, suggesting physical interactions between RF and cilia projecting into the central canal. We observed that motile cilia were caudally-tilted and frequently interacted with RF. We propose that the numerous ependymal motile monocilia contribute to RF's heterogenous tension via weak interactions. Our work demonstrates that under tension, the Reissner fiber dynamically interacts with motile cilia generating CSF flow and spinal sensory neurons.

Circadian clock1a coordinates neutrophil recruitment via nfe212a/duox-reactive oxygen species pathway in zebrafish

Neutrophil recruitment to inflammatory sites appears to be an evolutionarily conserved strategy to fight against exogenous insults. However, the rhythmic characteristics and underlying mechanisms of neutrophil migration on a 24-h timescale are largely unknown. Using the advantage of invivo imaging of zebrafish, this study explored how the circadian gene clock1a dynamically regulates the rhythmic recruitment of neutrophils to inflammatory challenges. We generated a clock1a mutant and found that neutrophil migration is significantly increased in caudal fin injury and lipopolysaccharide (LPS) injection. Transcriptome sequencing, chromatin immunoprecipitation (ChIP), and dual-luciferase reporting experiments suggest that the clock1a gene regulates neutrophil migration by coordinating the rhythmic expression of nfe212a and duox genes to control the reactive oxygen species (ROS) level. This study ultimately provides a visual model to expand the understanding of the rhythmic mechanisms of neutrophil recruitment on a circadian timescale in a diurnal organism from the perspective of ROS.

A novel 1,3,5-Triazine-based fluorescent probe with large Stokes shift for the detection of Hg(II) and its application in cell, zebrafish and tobacco imaging

Non-biodegradable, bioaccumulative, and highly toxic, mercury(II) poses a significant threat to both ecosystems and human health. Extensive data indicates that even extremely low levels of Hg(II) can cause significant harm to human health. In this paper, the thiocarbonate specific recognition site was used for the detection of Hg(II), which was successfully synthesized by the hydroxyl group of 1,3,5-Triazine with phenyl chlorothiocarbonate. The probe exhibited advantages of high sensitivity (0.253 μM), good selectivity, large Stokes shift (100 nm), and good biocompatibility. The probe TzAr-Nap-Hg successfully fluorescently imaged Hg(II) in cells, zebrafish and tobacco, making it possible to detect the content of Hg(II) in agricultural and sideline products.

Red fluorescent protein dimer for singlet oxygen (1O2)/ superoxide radical (O2•−) simultaneously mediated photodynamic therapy and two-photon fluorescence imaging

In this work, two lysosome-targeting red fluorescent protein analogues (APFP-lyso and DAPFP-lyso) were synthesized by phenolthiazine and anthracene anchoring strategies for singlet oxygen (1O2)/superoxide radical (O2•−) simultaneously mediated photodynamic therapy (PDT) in A-549 cells and two-photon fluorescence imaging in zebrafish. Compared with APFP-lyso of single-branch structure, fluorescent protein dimer DAPFP-lyso shows larger Stokes shift (Δλ = 5431 cm−1) with near-infrared emission at 664 nm and achieves 1.2- and 10.2-fold enhancement in 1O2 and O2•− production efficiency, respectively. In particular, effective two-photon absorption property of fluorescent protein dimer DAPFP-lyso (δ2PA = 81 GM) imparts the vivid two-photon fluorescence imaging in zebrafish under 800 nm excitation. Theoretical calculation indicated that fluorescent protein dimer DAPFP-lyso shows small singlet-triplet (ΔEST = 0.32 eV) to boost reactive oxygen species production efficiency. Intracellular PDT in A-549 cells, it is worth mentioning that DAPFP-lyso showed good biocompatibility, negligible cell dark toxicity (MTT assay >90%) and high phototoxicity (IC50 = 4.52 μM). The fluorescence imaging in A-549 cells and zebrafish manifests dimer DAPFP-lyso could generate 1O2 and O2•− under irradiation (460 nm, 23 mW cm−2). AO/EB dual staining assays and cell migration experiment in A-549 cells demonstrated that DAPFP-lyso could promote cancer cell apoptosis and migration under irradiation. The fluorescent protein dimer photosensitizer DAPFP-lyso had a precise lysosome-targeting ability (R = 0.95) could recognize lysosome specifically. These experimental results show that the fluorescent protein dimer design is of great significance for the development of new photosensitizers in the field of PDT.

NIR ditriphenylamine Indole-BODIPY photosensitizer: synthesis, photodynamic therapy in A549 cells and two-photon fluorescence imaging in zebrafish

In this study, the ditriphenylamine Indole-BODIPY photosensitizer T2BDP-lyso was synthesized for near-infrared photodynamic therapy and two-photon fluorescence imaging. The photosensitizer T2BDP-lyso exhibits absorption above 700 nm and emission above 800 nm, respectively. Theoretical calculations show the energy gap from the excited state S1 to the excited state T2 is 0.14 eV, which indicated that the photosensitizer T2BDP-lyso could reach the triplet state by intersystem crossing from the singlet state. Under NIR light, the singlet oxygen yield of photosensitizer T2BDP-lyso was calculated to be 0.64 in CH2Cl2. The photosensitizer T2BDP-lyso can effectively produce reactive oxygen species in A549 cells and zebrafish under 660 nm light for 5 min. The photosensitizer T2BDP-lyso exhibited lower dark toxicity and higher phototoxicity (IC50 = 1.49 μM), as well as lysosomal targeting ability (Pearson coefficient = 0.89). In the AO/EB double staining assay simulating photodynamic therapy at the cellular level, 3 μM of T2BDP-lyso light for 10 min was effective in killing cancer cells. Moreover, the photosensitizer T2BDP-lyso has a large two-photon absorption cross section at 1050 nm, which was calculated to be 138.7 GM in THF by Z-scan method, and two-photon fluorescence imaging was performed in zebrafish. The above results indicate the potential application of the photosensitizer T2BDP-lyso in near-infrared photodynamic therapy and two-photon fluorescence imaging.

Brain Imaging and Registration in Larval Zebrafish.

Registration of larval zebrafish brain scans to a common reference brain enables comparison of transgene and gene expression patterns, neuroanatomy, and morphometry. Here we describe methods for staining and mounting larval zebrafish to facilitate whole-brain fluorescence imaging. Following image acquisition, we provide a template for aligning brain images to a reference atlas using nonlinear registration with the ANTs software package.

Three-dimensional morphological and fluorescent imaging of zebrafish with a continuous-rotational light-sheet microscope

Light-sheet fluorescence microscopy (LSFM) has been widely used to image the three-dimensional (3D) structures and functions of various millimeter-size bio-specimen such as zebrafish. However, the sample adsorption and scattering cause shading of the light-sheet illumination, preventing the even 3D image of thick samples. Herein, we report a continuous-rotational light-sheet microscope (CR-LSM) that enables simultaneous 3D bright-field and fluorescence imaging. With a high-accuracy rotational stage, CR-LSM records the outline projections and the fluorescent images of the sample at multiple rotation angles. Then, 3D morphology and fluorescent structure were reconstructed with a developed algorithm. Using CR-LSM, zebrafish’s whole-fish contour and blood vessel structures were obtained simultaneously.

Unlocking the Potential of Zebrafish Research with Artificial Intelligence: Advancements in Tracking, Processing, and Visualization.

Zebrafish have become a widely accepted model organism for biomedical research due to their strong cortisol stress response, behavioral strain differences, and sensitivity to both drug treatments and predators. However, experimental zebrafish studies generate substantial data that must be analyzed through objective, accurate, and repeatable analysis methods. Recently, advancements in artificial intelligence (AI) have enabled automated tracking, image recognition, and data analysis, leading to more efficient and insightful investigations. In this review, we examine key AI applications in zebrafish research, including behavior analysis, genomics, and neuroscience. With the development of deep learning technology, AI algorithms have been used to precisely analyze and identify images of zebrafish, enabling automated testing and analysis. By applying AI algorithms in genomics research, researchers have elucidated the relationship between genes and biology, providing a better basis for the development of disease treatments and gene therapies. Additionally, the development of more effective neuroscience tools could help researchers better understand the complex neural networks in the zebrafish brain. In the future, further advancements in AI technology are expected to enable more extensive and in-depth medical research applications in zebrafish, improving our understanding of this important animal model. This review highlights the potential of AI technology in achieving the full potential of zebrafish research by enabling researchers to efficiently track, process, and visualize the outcomes of their experiments.

Construction of a novel chitosan-based macromolecular nanoprobe for specific fluorescent detection of H2S in live animals

H2S is one of the signal molecules in live organisms and a poisonous gas, which is closely related to our life. The traditional synthetic small molecular organic probes often have the disadvantages of low biocompatibility. In this paper, a fluorescent nanoprobe for detecting H2S in live organisms was constructed based on chitosan. The structure of CH-CN was characterized by infrared spectroscopy, nuclear magnetic resonance, x-ray photoelectron spectroscopy (XPS), XRD and scanning electron microscope (SEM). In the presence of Na2S, the fluorescence intensity at 560 nm was significantly enhanced, and showed high selectivity and sensitivity toward H2S. Based on the good fluorescence response of CH-CN, the probe was also successfully applied to H2S imaging in HepG2 cells and zebrafish. These experimental results indicate that the probe has lower cytotoxicity and excellent stability. The present research shows a typical example of construction of chitosan-based macromolecular fluorescent materials and their bio-imaging application.

Fluorescent protein chromophore-based photosensitizer modified with anthracenyl for lysosome-targeted photodynamic therapy and two-photon fluorescence imaging

In this work, two cis-anthracenyl fluorescent protein chromophore analogs, AnFP-1 and AnFP-2 were synthesized by [2 + 3] cycloaddition for photodynamic therapy and two-photon fluorescence imaging. We combine the anthracenyl group and fluorescent core imidazolinone to form the dye AnFP-1, and then the morpholine indole structure was introduced via Knoevenagel reaction to obtain the dye AnFP-2. The heavy-atom-free photosensitizing dyes AnFP-1 and AnFP-2 have satisfied yields of singlet oxygen, measured to be 30% and 21% in methanol solution, respectively. The tetrazolium blue (MTT) experiment showed that the survival rate of A549 cells was over 92% without light exposure and under 460 nm light exposure for 10 min, the half-inhibitory concentrations (IC50) of the photosensitizing dyes AnFP-1 and AnFP-2 were 3.7 μmol•L−1 and 4.1 μmol•L−1, respectively. Acridine orange (AO)/Ethidium bromide (EB) double staining experiments showed that the two photosensitizing dyes could generate singlet oxygen and induce apoptosis in cancerous cells under irradiation. In addition, the photosensitizing dye AnFP-2 has high specificity to lysosomes which improve the photodynamic effect. Its Pearson co-localization correlation coefficient is 93%. In biological imaging, AnFP-1 and AnFP-2 showed satisfactory two-photon fluorescence imaging in zebrafish with 800 nm femtosecond laser excitation. According to these results, AnFP-1 and AnFP-2 can be used in photodynamic therapy and two-photon fluorescence imaging, which lays the foundation for developing new photosensitizing dyes.

Deep-MSIM: Fast Image Reconstruction with Deep Learning in Multifocal Structured Illumination Microscopy.

Fast and precise reconstruction algorithm is desired for for multifocal structured illumination microscopy (MSIM) to obtain the super-resolution image. This work proposes a deep convolutional neural network (CNN) to learn a direct mapping from raw MSIM images to super-resolution image, which takes advantage of the computational advances of deep learning to accelerate the reconstruction. The method is validated on diverse biological structures and in vivo imaging of zebrafish at a depth of 100µm. The results show that high-quality, super-resolution images can be reconstructed in one-third of the runtime consumed by conventional MSIM method, without compromising spatial resolution. Last but not least, a fourfold reduction in the number of raw images required for reconstruction is achieved by using the same network architecture, yet with different training data.

Visible-light Excited and Highly Photostable Organic Fluorophore with dual-state Emission.

Organic fluorophores with dual-state emission (DSE) are rare or difficult to observe because most of them display either aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ). Amazing works have been accomplished, yet most of the DSE compounds were excited by UV light which limits their wide application in bioimaging. In this work, we achieved a visible-light excited DSE fluorophore and realized its imaging in SKOV-3 cells and zebrafish. The naphtho[2',3':4,5]imidazo[1,2-a]pyridine (NIP) core ensures its emission in dilute solution. Meanwhile, the twisted phenyl ring blocks fluorescence quenching induced by the π-π stacking and leads to the emission of the solid. The fluorescence intensity is steady even after 6h of continuous intense sunlight. More importantly, photostability of NIP in cells is much better than commercial dye (mitochondrial green).