Zebrafish-Based Bioimaging: In Vivo Visualization of Drug Effects at Organ Level

Modern pipelines need faster ways to see how compounds act across organs in vivo, before committing to slow and costly mammalian studies. Zebrafish enable whole-organism imaging with cellular resolution, fast timelines, and high-throughput. When combined with automated bioimage acquisition and analysis, they deliver robust, quantitative evidence on efficacy and safety, saving time, money, and animals. 

What Is Bioimaging and Why Use Zebrafish for It?

Bioimaging is the use of imaging technologies to visualize biological structures and processes in living systems, to measure, quantify, and understand their behavior.  It combines hardware (microscopes, scanners), probes (e.g., fluorescent proteins, dyes, nanoparticles), and computational analysis to turn images into data about morphology, dynamics, and function. Bioimaging makes it possible to track development, disease progression, drug responses, and signaling events in vivo and is now central to developmental biology, drug discovery, disease progression monitoring, and toxicological assessment. 

Zebrafish is a representative organism for bioimaging. Their embryos and larvae are optically transparent, which allows direct visualization of internal organs, cells, and even subcellular structures in vivo. A rich toolbox of transgenic lines and genome editing allows tissue‑specific fluorescent reporters and disease‑relevant knock‑in/knock‑out models, which can then be followed by fluorescent bioimaging to link genotype, cellular phenotype, and organism‑level outcomes. Besides, they are small and highly fecund, which enables multi-well plate workflows.

These advantages position zebrafish as a strong fit for high-throughput discovery and toxicology screens. However, no platform reaches true scale without automation. 

VAST BioImager in High-Throughput Zebrafish Imaging

High-throughput imaging depends on repeatable positioning and orientation of zebrafish larvae. The VAST BioImager coupled to the Large Particle sampler and dispenser automates loading, alignment, and multi-angle imaging for large cohorts of 2-7 days post-fertilization (dpf) larvae. It provides brightfield and fluorescence images at fine spatial resolution and enables 3D optical projection tomography when needed. 

Figure 1. VAST BioImager is a modular and expandable platform. It can be mounted on many microscopes for high-resolution and fluorescence imaging.

The VAST BioImager supports imaging at whole‑fish and organ‑level resolution (around 10 µm). When coupled to a fluorescence‑equipped microscope, it becomes a high‑content platform for imaging transgenic reporter lines, allowing dynamic, in vivo monitoring of organ‑specific signals across large cohorts. 

By automating loading, positioning, orientation, and ejection/dispensing of each larva, the system eliminates the most tedious and time‑consuming manual steps of zebrafish imaging workflows. It also reduces operator variability and increases statistical power for screening.

Analyzing VAST images with deep learning adds another layer of precision. It improves segmentation of specific regions (e.g., head, eye, heart) and enables unbiased extraction of morphometric and functional endpoints at scale. A great example is the extraction of developmental defects in embryos exposed to different toxic compounds to identify teratogenic phenotypes. At ZeClinics, we perform this analysis as part of our developmental toxicity assessment (Figure 2). 

Figure 2. Extraction of teratogenic phenotypes in 120-hour post-fertilization (hpf) zebrafish larvae images obtained with VAST BioImager after exposure to a teratogenic compound. 

Applications in Organ-Level Visualization: Brain, Heart, Liver, and More

Zebrafish larvae and automated imaging make it straightforward to observe how drugs affect organs in real time. By 5 dpf,  major systems are functional, allowing short imaging sessions to capture brain activity, heart performance, and liver health under controlled conditions. The result is clear, quantifiable readouts that link dose to organ response in the same organism. Some bioimaging examples at the organ-level include: 

• Brain. By 5 dpf, larvae display robust visual-motor behavior. Neurofunctional effects are captured as changes in total distance moved under light-dark cycles, using plate-based video tracking. Hyperactivity and hypoactivity patterns distinguish central excitatory from depressant actions and can be linked to specific compounds at defined doses.  

• Heart. From 96 to 100 hpf, stabilized cardiac rhythms allow imaging-based quantification. High-speed videos of fluorescent hearts yield beats per minute, QTc interval, ejection fraction, and arrest duration. We have demonstrated that reference drugs produce the expected signatures: haloperidol and pindolol prolong QTc, while ciprofloxacin and glucose shorten it, allowing early detection of rhythm liabilities in vivo (Cornet et al. 2017).
• Liver. Fluorescent liver reporters and specific staining, such as Oil Red O, allow to evaluate different endpoints to detect hepatotoxicity based on image analysis (Figure 3). These endpoints include: liver area for hepatomegaly or cell loss, steatosis, and yolk lipid retention. 

Figure 3. Representative images of healthy zebrafish larva (control -), steatotic zebrafish liver (control +), and induced steatotic zebrafish by a candidate molecule (compound 1). 

These examples show how standardized imaging, deep-learning segmentation, and predefined endpoints help convert organ-level phenotypes into robust, decision-ready features suitable for early go/no-go. 

Advancing Drug Discovery Through In Vivo Fluorescent Reporters and Imaging Assays

Fluorescent reporters in zebrafish turn pathway activity and organ biology into measurable screening readouts. Several examples in the literature show how these lines enable compound discovery with clear, image-based endpoints.

In pathway modulation screens, the FGF-responsive line Tg(dusp6:EGFP) visualizes FGF activity in vivo and is readily sensitized by pharmacological antagonists, making it useful for identifying small molecules that up- or down-modulate FGF signaling during development. Reporter intensity changes provide a direct, quantitative assay for hit identification.

In the field of renal regeneration and nephroprotection, the Tg(wt1b:eGFP) line has contributed to the research on the development and regeneration of the kidney, as well as the drug screening against nephrotoxicity and genetic kidney diseases, including hereditary glomerulopathies and cystic kidney diseases. 

Oncology has also witnessed great advances in drug discovery thanks to fluorescent reporter lines. 

A panel of 4,880 bioactive small molecules was screened for the ability to kill fluorescently labelled T lymphocytes in larval transgenic zebrafish. The analysis identified a lead compound, phenothiazine (PPZ), which also killed T cells in MYC-induced T cell acute lymphoblastic leukaemia (T-ALL) transgenic zebrafish models and human T-ALL cell lines (Gutierrez et al. 2014).

At ZeClinics, we have the expertise and the capabilities to generate any custom transgenic model to exploit zebrafish transparency and potential. We can introduce genetic reporters to visualize specific tissues or cell types to monitor biological processes of interest in a time-specific manner. Our lab is equipped with a VAST BioImager, which allows us to perform high-throughput phenotype screenings. 

You just need to ask the scientific question, and we will be here to answer it, controlling the process from model generation to high-throughput imaging, quantitative analysis, and decision-ready reporting. 

Ask the question!

Source

Choe CP, Choi SY, Kee Y, Kim MJ, Kim SH, Lee Y, Park HC, Ro H. Transgenic fluorescent zebrafish lines that have revolutionized biomedical research. Lab Anim Res. 2021 Sep 8;37(1):26. doi: 10.1186/s42826-021-00103-2.

Cornet C, Calzolari S, Miñana-Prieto R, Dyballa S, van Doornmalen E, Rutjes H, Savy T, D'Amico D, Terriente J. ZeGlobalTox: An Innovative Approach to Address Organ Drug Toxicity Using Zebrafish. Int J Mol Sci. 2017 Apr 19;18(4):864. doi: 10.3390/ijms18040864.

Gutierrez A, Pan L, Groen RW, Baleydier F, Kentsis A, Marineau J, Grebliunaite R, Kozakewich E, Reed C, Pflumio F, Poglio S, Uzan B, Clemons P, VerPlank L, An F, Burbank J, Norton S, Tolliday N, Steen H, Weng AP, Yuan H, Bradner JE, Mitsiades C, Look AT, Aster JC. Phenothiazines induce PP2A-mediated apoptosis in T cell acute lymphoblastic leukemia. J Clin Invest. 2014 Feb;124(2):644-55. doi: 10.1172/JCI65093.

Patton EE, Zon LI, Langenau DM. Zebrafish disease models in drug discovery: from preclinical modelling to clinical trials. Nat Rev Drug Discov. 2021 Aug;20(8):611-628. doi: 10.1038/s41573-021-00210-8. 

Union Biometrica. VAST BioImager platform [Internet]. Union Biometrica; [cited 2025 Dec 18]. Available from: https://www.unionbio.com/vast/bioimager-platform.aspx

By Miriam Martínez

Miriam is a Human Biologist with a strong background in neuropharmacology and a passion for bridging science and innovation. After earning a master’s degree in the Pharmaceutical and Biotech Industry, she completed her PhD in Biomedicine at Pompeu Fabra University (Barcelona), where her research focused on the behavioral analysis of animal models for neurophenotypical characterization. Following her doctoral studies, Miriam transitioned into the healthcare marketing and communication sector, where she played a key role in developing impactful marketing strategies and educational campaigns for leading pharmaceutical brands. She now leverages her scientific expertise, strategic thinking, and creative communication skills in her current role at ZeClinics.