Background

Zebrafish (Danio rerio) have become a widely popular vertebrate model organism given their early larval transparency, highly conserved genome with humans, external embryo fertilization, and rapid development compared to other vertebrates. These features position zebrafish as a well-suited model for in vivo observations across fields like developmental biology, neuroscience, genetics, and toxicology [1,2]. This has enabled their use as a robust predictive model for teratogenicity, with morphometric profiling of development offering insights into chemical-specific consequences and modes of action conserved across vertebrates [3]. More broadly, many modern experimental paradigms integrate different imaging modalities with genetic manipulations and behavioral assays to both influence and capture biological dynamics with larval zebrafish in real time [4]. However, fully exploiting these capabilities with non-invasive imaging techniques often depends on mounting orientation and optical accessibility.

These practical constraints become particularly important when selecting optical imaging modalities that differ in penetration depth, speed, and configuration. Techniques like calcium imaging, photochemical conversion of fluorophores via targeted stimulation, optogenetics, and photoinduced ablation benefit from the advantages of multiphoton microscopy, including deeper penetration than in linear techniques like confocal imaging [5]. A great example involves the increased engineering of in vivo indicators to exploit the nonlinear physics of multiphoton excitation. Recent advancements have leveraged rhodopsin and calcium-based sensors to establish genetically encoded calcium (GECI) and voltage (GEVI) indicators across wavelengths that maximize photon capture and signal-to-noise ratio in deep tissue [6,7]. On the other hand, the faster imaging speeds, greater ease of setup, and robust optical sectioning provided by linear microscopes, specifically confocal systems, are especially useful when imaging rapid biological dynamics where temporal resolution and precision in optical sectioning are required [8]. Combining both approaches can integrate the depth of multiphoton penetration while maintaining the rapid visualization and temporal resolution provided by confocal imaging, but the ease of use with larval embryos can be limited by opposing microscope objective orientations. Though varying modalities can be integrated into a single system, like the addition of a photostimulation laser to a spinning-disk confocal system, doing so often entails substantial cost and complexity.

Traditional lateral mounting for inverted imaging positions larval zebrafish against the bottom of a glass-bottom imaging dish, secured with a mound of agarose. This configuration allows for clear visibility of anterior–posterior axis development in zebrafish and helps to standardize anatomical positioning across samples. However, because the imaging dish is filled with liquid and embryos are fixed against the bottom of the dish, optimal accessibility is limited to imaging through the glass from below. Observation from above, as with top-down microscopes, is constrained by working distance limitations and obstruction from the mounting agar and anesthetic solution. This restricts flexibility across microscope geometries, as samples can only be imaged with the shortest optical distance from one side. Additionally, direct water immersion with the upright objective into the imaging dish risks damage to samples and mounting. The optical distance can be extended to target the region of interest across configurations without remounting, but this increases the limitations of multiphoton imaging in photobleaching, light scattering, and heat exposure [8]. Further, remounting samples can introduce variability in anatomical positioning, increase the required handling of anesthetized samples, and complicate the identification of the same biological region of interest for longitudinal assessment.

Here, we present a dual-configuration mounting model that enables easy transition across upright and inverted microscope geometries. This setup combines standard zebrafish mounting with a model constructed from common low-cost laboratory consumables to eliminate remounting across microscope configurations, supporting both upright and inverted imaging. Using a simple petroleum jelly assembly, we combine a 35 mm glass-bottom confocal imaging dish with a 60 mm Petri dish glued onto a microscope slide. This method successfully preserves the conventional lateral embryo positioning while enabling optical access from both upright and inverted perspectives to allow for rapid transition between imaging geometries. Ultimately, this configuration supports workflows that combine targeted interventions with long-term imaging and can be applied using any mounting orientation.