Introduction

Why is Volumetric Imaging Important?

Biological processes occur in a fundamentally three-dimensional (3D) space, and the spatial organization of molecular structures is critical to cellular function. Traditional two-dimensional (2D) imaging provides only a limited cross-section of these dynamic events, often obscuring key interactions and leading to incomplete or misleading interpretations.

For instance, during mitosis, chromosomes undergo intricate rotations within the entire cellular volume, and microtubules dynamically polymerize and depolymerize as they engage and separate daughter chromosomes at their kinetochores. In 2D imaging, these structures inevitably drift in and out of the focal plane, making it challenging to track such dynamic processes over time. Similarly, in complex tissue environments, organelle positioning, cell-cell interactions, and subcellular signaling pathways are all inherently shaped by their 3D spatial context.

By capturing the full volume of a specimen, volumetric imaging enables a more accurate representation of cellular architecture and dynamic molecular events. This is particularly critical for understanding developmental biology, disease progression, and cellular interactions within their native environments. Emerging technologies such as light-sheet microscopy, expansion microscopy, and advanced computational imaging now allow researchers to visualize biological systems at unprecedented resolutions, bridging the gap between molecular detail and tissue-scale organization.

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The Importance of Parallelization

Historically, 3D imaging has been performed using spinning disk and laser scanning confocal microscopes. However, these microscopes waste significant excitation energy on out-of-focus regions, leading to unnecessary photodamage while requiring compensatory increases in laser power due to their low illumination duty cycle. This problem is further exacerbated by their sequential, point-by-point image acquisition, which limits speed and increases photobleaching.

Illumination Confinement

Illumination confinement refers to the restriction of excitation light to within the depth of focus of the detection objective, ensuring that only light contributing to image formation is used. This is a key advantage of light-sheet fluorescence microscopy (LSFM), where the illumination plane is matched to the detection plane, minimizing out-of-focus excitation and reducing phototoxicity.

In contrast, depending on their design, light-sheet fluorescence microscopes (LSFM) restrict illumination primarily to the focal plane of interest, minimizing phototoxicity while maximizing imaging efficiency. Additionally, by leveraging highly sensitive scientific cameras (e.g., 4×10⁶ pixels), LSFM enables massively parallelized image acquisition—recording entire planes in a single exposure rather than scanning point-by-point.

For instance, a typical laser scanning confocal microscope requires ~4.16s to acquire a 2048 × 2048 voxel image, given a voxel dwell time of 1µs. In contrast, an LSFM can image the same region in just ~5ms, an 832-fold increase in speed, all while maintaining a 5,000-fold longer per-voxel dwell time. This extended dwell time enables improved signal accumulation, lower laser power requirements, and a significantly improved signal-to-noise ratio (SNR). As a result, LSFM is uniquely suited for long-term volumetric imaging at high spatiotemporal resolution while reducing photodamage.

Spatial Duty Cycle

The spatial duty cycle of illumination refers to the fraction of a sample that is actively contributing to image formation at any given moment during an imaging process. In laser scanning confocal microscopy, only a single point is illuminated at a time, resulting in a low duty cycle and requiring compensatory increases in laser power to maintain signal. In contrast, light-sheet fluorescence microscopy (LSFM) illuminates and images an entire plane at once, yielding a duty cycle that approaches 100%, enabling drastically reduced laser powers that reduce phototoxicity, .

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Challenges with 3D Imaging

To extract meaningful biological insight from volumetric imaging, microscopes must achieve sufficient spatiotemporal resolution while preserving molecular specificity and compatibility with advanced analytical techniques. This requires integration with biosensors, opto- and chemogenetic tools, and computer vision analyses that translate high-dimensional 5D datasets (x, y, z, \(\lambda\), t) into quantitative biological readouts. Several key challenges must be addressed to ensure the accuracy and reliability of 3D imaging:

• Nyquist Sampling in Space and Time: To faithfully capture dynamic processes, the event of interest must be Nyquist sampled in both spatial and temporal dimensions. For example, the GTPase cycle times of Rho, Rac, and Cdc42 can be as short as 5s, necessitating volumetric acquisitions at \(\leq\) 2.5s per volume with a spatial resolution of <500nm. Furthermore, resolution should ideally be isotropic or nearly isotropic to prevent morphology-dependent intensity artifacts—particularly for signaling events at the plasma membrane.

• Multicolor Excitation and Detection: To enable multiplexed cellular readouts, the microscope must support simultaneous multicolor imaging with achromatic performance across excitation and detection wavelengths. Microscope designs that rely on spatial light modulators (SLMs) for excitation often struggle to achieve this, as the diffraction efficiency of SLMs varies with wavelength.

• Detection Sensitivity and Phototoxicity: Imaging performance must be photon-efficient to minimize the need for high ectopic expression of signaling-active proteins and to mitigate photobleaching and phototoxicity—a challenge that is amplified when acquiring 10–100 slices per volume. Maximizing quantum efficiency and minimizing out-of-focus excitation are crucial for maintaining live-cell viability.

• Field of View Constraints: When imaging dynamic processes in extracellular matrix environments (where cells can migrate in any direction) or within a developing embryo, the microscope must provide a sufficiently large field of view (FOV)—ideally >100 × 100 × 100 µm. Many leading light-sheet fluorescence microscopes (LSFMs) are optimized for small fields of view (e.g., 25µm), which limits their applicability to studies of large-scale tissue dynamics.

• Avoidance of Computational Post-Processing Biases: To ensure compatibility with quantitative downstream analyses, reliance on iterative deconvolution or structured illumination routines should be minimized. These techniques can introduce statistical artifacts that distort numerical analyses, particularly when measuring intensity distributions, localization precision, or dynamic molecular interactions.

Nyquist Sampling

Nyquist sampling is a fundamental principle in signal processing that dictates the minimum sampling frequency required to accurately reconstruct a signal without aliasing. According to the Nyquist-Shannon sampling theorem, a continuous signal must be sampled at least twice the highest frequency component present in the signal to ensure faithful reconstruction.

In microscopy, Nyquist sampling applies to both spatial and temporal domains:

• Spatial Nyquist Sampling: To accurately resolve features of size \(d\), the sampling interval (pixel or voxel spacing) should be no greater than \(d/2\). This ensures that high-frequency structural details are captured without loss of information.

• Temporal Nyquist Sampling: To track dynamic processes occurring at a characteristic frequency \(f_c\), images must be acquired at a frequency of \(\geq 2 \times f_c\) to prevent aliasing in time-lapse imaging.

Failure to meet the Nyquist criterion results in aliasing, where high-frequency components are misrepresented as lower-frequency artifacts, distorting biological measurements.

Meeting these criteria is essential for accurate, high-throughput volumetric imaging that captures cellular dynamics with sufficient fidelity to support rigorous biological interpretation.

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Why Build a Microscope?

The technology required to achieve multiplexed volumetric imaging with advanced probes and computer vision analyses already exists. However, the commercialization process imposes significant constraints on innovation. Microscope manufacturers prioritize aesthetically attractive, highly engineered, and serviceable optical systems that ensure a large return on investment. As a result, they tend to be extremely conservative in adopting emerging technologies.

Most commercially available microscopes take over seven years to develop, by which time they are often already obsolete due to rapid scientific advancements. A notable exception is the Lattice Light-Sheet Microscope (LLSM), which was sublicensed by Zeiss to 3i shortly after its seminal publication. However, even in this case, it took another six years for Zeiss to release a consumer-friendly model—at a prohibitive cost of ~$1M USD.

Beyond commercialization delays, patent restrictions further stifle innovation. The highly fragmented and entangled intellectual property (IP) landscape makes it difficult for new start-ups to enter the market. For example, despite their own limited role in developing oblique plane microscopy (OPM), Leica has exclusively licensed a patent for off-axis tertiary imaging systems, effectively blocking broader commercialization of OPM.

As a result, reliance on commercial microscope development not only delays technology adoption but can actively impede the dissemination of transformative imaging methods. This reality makes in-house development of custom microscopy platforms essential for pushing the frontiers of biological imaging forward.