Organ-scale spatial phenotyping at subcellular resolution

The Challenge: Bridging the scale-resolution gap while preserving native tissue integrity

A comprehensive understanding of biological systems demands the visualization of complex tissue architecture in 3D without sacrificing subcellular detail. However, current spatial biology methods face a critical trade-off:

Traditional 2D Histology: Offers high resolution but severs volumetric connectivity.
3D Tissue Clearing: Provides volumetric data but compromises molecular integrity and requires time-intensive chemical processing.

Our Solution:

We are advancing high-content volumetric tomography to bridge the gap between organ-scale coverage and subcellular resolution. Our automated platforms enable the systemic analysis of intact biological networks in their native state.

Core technologies

Translational rapid ultraviolet-excited sectioning tomography (TRUST)

Figure 1. System setup and workflow of the TRUST system.

Figure 2. TRUST images of mouse organs and embryos.

We established TRUST as a platform capable of imaging whole organs (e.g., brain, lung, spleen) and embryos without clearing, prestaining or dehydration.

Mechanism: Utilizes deep-UV surface excitation and automated real-time staining.
Advantage: Preserves native tissue geometry and molecular integrity.
Speed: Whole-embryo staining and imaging completed within 2 days.

HistoTRUST: Virtual 3D histology assisted with deep learning

Figure 3. Principle and workflow of HistoTRUST

Bridging 3D TRUST imaging with 3D pathology
To integrate our 3D data with standard clinical workflows, we developed HistoTRUST.

Mechanism: We employ generative models to perform modality transfer, converting label-free or fluorescence signals into virtual H&E standards.
Impact: This facilitates direct cross-modality analysis and allows pathologists to interpret 3D volumetric data using traditional diagnostic criteria.

HiLoTRUST: Optical sectioning with improved axial resolution

Figure 4. System setup and results of the HiLoTRUST system.

For standard widefield microscopy, imaging axial resolution and contrast can considerably deteriorate when the light penetration depth is significantly larger than the objective lens’s depth of field. This can occur when imaging tissue with relatively low scattering properties (e.g., human breast) or when using a high-magnification objective lens. To address the issue, we present HiLoTRUST, which integrates the high-and-low-frequency (HiLo) microscopy into TRUST for better optical sectioning capability and imaging contrast by rejecting the out-of-focus fluorescence background.

Mechanism: Integrates Speckle Illumination with UV excitation to actively reject out-of-focus background signals.
Result: Improves axial resolution to ~5.8 µm, enabling high-contrast 3D reconstruction of dense tissue structures that standard methods cannot resolve.

Ongoing directions

Moving beyond structural mapping, our ongoing research expands these platforms to achieve higher spatial and spectral precision. On the spatial front, we are developing lightsheet tomography to realize whole-organ imaging with isotropic sub-micron resolution. Simultaneously, we are engineering a deep-UV excited hyperspectral microscopy system to profile molecular composition. By applying spectral unmixing to these endogenous autofluorescence signals, we aim to decode native metabolic states, pushing the boundaries of label-free spatial phenotyping.

References

Yu, W. et al. Translational rapid ultraviolet-excited sectioning tomography for whole-organ multicolor imaging with real-time molecular staining. eLife 11, 1–21 (2022).
Kang, L., Yu, W., Zhang, Y., Chen, Z. & Wong, T. T. W. Deep Learning Enables Rapid Whole-Organ Histological Imaging with Ultraviolet-Excited Sectioning Tomography. ACS Photonics 10, 3541–3550 (2023).
Yu, W., Zhang, Y., Lo, C. T. K., Kang, L. & Wong, T. T. W. Rapid full-color serial sectioning tomography with speckle illumination and ultraviolet excitation. npj Imaging 2, 37 (2024).

Engineering protein-bound pigments for imaging-guided therapy

Our laboratory develops biocompatible protein-based contrast agents for advanced photoacoustic (PA) imaging applications. We engineer water-soluble chlorophyll-binding proteins (WSCP) from plants to create molecular-specific imaging agents that target cancer biomarkers like HER2 found in breast cancer. Beyond imaging, we're expanding into therapeutic applications including photodynamic therapy, creating multifunctional biomaterials that bridge diagnostics and treatment. Our goal is to translate these technologies into clinical applications for improved cancer detection and image-guided interventions.

Low-Cost Deep Tissue Imaging

While lasers have been commonly used as illumination sources in photoacoustic imaging, their high purchase and maintenance costs, as well as their bulkiness, have hindered the rapid clinical dissemination of photoacoustic imaging. With this in mind, we explore an alternative illumination source for photoacoustic tomography — a xenon flash lamp with high pulse energy and a micro-second pulse width. We demonstrate that, by using a single xenon flash lamp, we can image both a black latex cord placed in chicken breast tissue at a depth of up to 3.5 cm ex vivo, and an entire mouse body in vivo. Our findings indicate that the xenon flash lamp, producing optical illumination that is safe for humans, can be potentially applied to human tissue imaging.

Reference:

Terence T. W. Wong, Yong Zhou, Alejandro Garcia-Uribe, Lei Li, Konstantin Maslov, Li Lin, Lihong V. Wang, "Use of a single xenon flash lamp for photoacoustic computed tomography of multiple-centimeter-thick biological tissue ex vivo and a whole mouse body in vivo," J. Biomed. Opt. 22(4), 041003 (2016) [Link]