Challenges of Imaging In Vitro Models; Part 1: Field of View

By Parisa Iloun, Apr 2026

In the field of neuroscience, biological samples encompass a wide range of structures. These include spheroids, organoids, and assembloids, as well as in vivo models such as zebrafish, C. elegans, and Drosophila embryos, and in vitro preparations like whole-brain slices from rodents, among others. In experimental settings, imaging these samples in their entirety at cellular resolution can help address key questions, for instance, to investigate functional processes such as neuronal network dysfunction in disease models or synchronized activity across interconnected networks. Whole-structure imaging is also essential for studying developmental dynamics, including cell migration and the growth of axonal projections as interconnected organoids to establish functional neural circuits.

However, capturing the full structure of these models remains challenging, primarily due to their relatively large size. Spheroids typically range from 100–500 µm, organoids from 0.5–3 mm, and assembloids can reach 1–4 mm or more. Whole organisms such as zebrafish larvae and brain slices can also span several millimeters. In contrast, the field of view (FOV) of many commonly used microscopy techniques is either limited to only a few hundred micrometers or at larger FOV they cannot provide single cell resolution. Therefore, there is a trade-off between resolution and FOV, with many microscopes technique optimizing for one or the other, without being able to provide a large FOV with cellular resolution.

Left: Image of assembloid, acquired with VoluScanTM,  Right: individual neurons identified and cellular traces extracted with danseTM analysis software

For instance, two-photon microscopy is optimized for high spatial resolution and deep tissue imaging [2]. It enables visualization of subcellular structures and can penetrate deeper into scattering tissue compared to conventional fluorescence microscopy. However, this comes at the cost of a limited field of view, making it less suitable for imaging cellular dynamics across a large structure.

Meanwhile, regular epifluorescence microscopes have the opposite challenge, where moderate samples <2.5mm require lower magnification objective to image its entirety, which typically reduces the optical resolution and the signal collection. Moreover, these types of microscopes cannot image large samples (2.5 to 5 mm).

In contrast, Light-sheet microscopy offers an alternative approach by enabling fast volumetric imaging and relatively large fields of view [3]. It is particularly well-suited for imaging of entire organoids, assembloids or zebrafish brains. However, these systems are often complex, require specialized sample preparation, and can be prohibitively expensive, limiting their accessibility for many laboratories.

There is therefore a need for imaging solutions that provide millimeter-scale fields of view while preserving cellular resolution, at an accessible cost. To address this gap, at Doric Lenses, we developed an advanced microscopy system: the one-photon, epifluorescence VoluScan™ microscope.

VoluScan™ system can image large, millimeter-scale samples (up to ~5x5 mm FOV, ~twice larger than regular fluorescence microscopes) while maintaining cellular resolution (resolution of ~2.7 µm) and depth coverage up to ~150 µm. This makes it particularly valuable for applications such as tracking cell migration, population dynamics, or large-scale activity patterns across entire samples [5].

Comparing relative size of samples and VoluScan™ FOV (blue boxes)

Meanwhile for a smaller sample, less than 2.5 mm, VoluScan™ can be paired with a higher magnification objective lens (twice higher than a standard microscope), doubling the optical resolution and increasing (~4 times) the fluorescence collection. A comparison of VoluScan and a conventional epifluorescent microscope in terms of field of view (FOV) and resolution using different objectives is shown below:

A compelling example of VoluScan™ applied to large-scale assembloid imaging comes from the laboratory of Sergiu P. Pașca at Stanford University. In a recent study, Miura et al. (2024, bioRxiv) imaged a large assembloid (~2 × 2 mm²) composed of four interconnected organoids in its entirety. Using VoluScan™, the authors recorded live-cell calcium activity (GCaMP8s) from over 400 cells within a single assembloid and demonstrated a functional model of the cortico striatal–thalamic–cortical circuit. The cellular resolution achieved enabled the validation of coordinated neural activity across the four organoid regions [5].

Modified image adapted from Miura et al. (2024, bioRxiv)

Additionally, to meet the needs of biological imaging applications, VoluScan™ is designed to image a wide range of fluorescent sensors, including GCaMP, jRGECO, dLight, and others. The system can also be readily integrated with additional experimental modalities such as light sources (for optogenetics or uncaging), electrophysiological recordings, and liquid delivery systems.

Furthermore, VoluScan™ is compatible with environmental control systems such as the Tokai Hit incubation chamber, enabling long term stable live-cell imaging conditions. The second generation VoluScan™ (Gen2) is equipped with a motorized 3D stage, allowing sequential imaging of multiple regions across large sample formats, including well plates and Petri dishes. By defining regions of interest in advance, users can perform automated, high-throughput imaging experiments, such as overnight acquisitions—significantly accelerating data collection.

References:

1. Clevers, H. (2016). Modeling development and disease with organoids. Cell.
2. Lancaster, M. A., & Knoblich, J. A. (2014). Organogenesis in a dish: modeling development and disease using organoid technologies. Science.
3. Denk, W. et al. (1990). Two-photon laser scanning fluorescence microscopy. Science.
4. Ahrens, M. B. et al. (2013). Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nature Methods.
5. Miura, Y. et al. (2024). Assembloid model to study loop circuits of the human nervous system. BioRxiv