Academic Background

Three-dimensional (3D) high-resolution large-volume imaging has remained a significant challenge in the field of biomedical research. Traditional two-dimensional (2D) slice imaging, while capable of providing planar morphological information of tissues and cells, fails to comprehensively display internal 3D structural information, which is crucial for cancer diagnosis and embryonic development studies. Conventional 3D histological methods typically require manual sectioning and staining of thousands of thin slices, which is time-consuming and labor-intensive. Additionally, complex image registration algorithms are needed to restore spatial information between different slices. To address these issues, various automated 3D optical imaging techniques have emerged in recent years, primarily divided into two categories: one involves tissue clearing techniques to reduce light propagation issues in biological tissues, while the other relies on block-face serial sectioning tomography (BSST) to expand imaging volume.

However, existing 3D imaging technologies still have some limitations. For example, tissue clearing techniques must balance clearing effects and time to prevent tissue degradation, while BSST systems, although capable of generating aligned images, are highly complex, especially in multicolor imaging. Moreover, many BSST systems require whole-mount staining and embedding samples in hard materials (such as resin or paraffin), which not only increases time and cost but also causes tissue dehydration and shrinkage.

To address these challenges, a team from the Hong Kong University of Science and Technology, including Wentao Yu, Yan Zhang, Claudia T. K. Lo, Lei Kang, and Terence T. W. Wong, developed a novel 3D imaging technology called HILOTRUST. This technique combines high-and-low-frequency (HILO) microscopy with ultraviolet (UV) excitation, enabling rapid, cost-effective, high-resolution whole-organ subcellular imaging.

Research Content and Workflow

Research Background and Objectives

The HILOTRUST technology is an optimization of the team’s previously developed TRUST (Translational Rapid Ultraviolet-Excited Sectioning Tomography) technique. TRUST achieves rapid, high-resolution whole-organ imaging through UV surface excitation and real-time staining, but its axial resolution is limited by the mechanical sectioning thickness or UV light penetration depth in tissues. To overcome these limitations, HILOTRUST introduces HILO microscopy, reducing the optical sectioning thickness from tens of micrometers to approximately 5.8 micrometers and the mechanical sectioning thickness from 50 micrometers to 10-15 micrometers.

Experimental Design and Methods

1. Setup of Full-Color HILO Microscopy

The core of the HILOTRUST system is the full-color HILO microscope. This microscope performs imaging through two illumination modes: speckle illumination and uniform illumination. In speckle illumination mode, a 532-nanometer green laser diode (LD) generates a speckle pattern to excite fluorescence signals in cell nuclei; in uniform illumination mode, two deep UV LEDs (UV-LEDs) provide uniform illumination to excite fluorescence signals from labeled dyes and endogenous tissue components. By combining these two modes, HILOTRUST achieves full-color imaging and significantly improves image contrast.

2. Setup of the HILOTRUST System

The HILOTRUST system integrates the full-color HILO microscope with a three-axis motorized stage and a vibratome. During imaging, the sample is first raster-scanned in the X-Y plane via the motorized stage, followed by mechanical sectioning with the vibratome. After each imaging session, the system automatically removes the imaged tissue layer to expose the next layer for imaging. The entire process is fully automated, requiring no manual calibration.

3. Data Processing and 3D Reconstruction

The HILOTRUST system generates optical sectioning images through the HILO image processing algorithm and performs 3D reconstruction via direct image stacking. Since the generated image slices are inherently aligned, no complex image registration algorithms are required.

Experimental Results

1. Validation of Full-Color HILO Microscopy

The research team first validated the high-content and optical sectioning capabilities of the full-color HILO microscope. By performing 2D imaging on various mouse organs (such as lung, kidney, liver, and brain), they found that HILO images significantly improved contrast compared to traditional widefield images, enabling clearer identification of fine structures.

2. 3D Imaging Validation

The team further conducted 3D imaging on mouse kidney, lung, and liver tissue blocks and compared the results with those obtained using TRUST technology. The results showed that HILOTRUST has a significant advantage in optical sectioning capability, generating clearer and more detailed 3D images. Particularly in the 3D reconstruction of mouse lung tissue, HILOTRUST was able to more accurately display the distribution of alveoli and elastic fibers.

3. Potential for Clinical Applications

To validate the potential of HILOTRUST in clinical applications, the team imaged two human lung cancer samples. The results demonstrated that HILOTRUST significantly improved image contrast, making structures such as elastic fibers easier to identify. Additionally, the full-color imaging capability of HILOTRUST allowed for the extraction of the 3D distribution relationship between collagen fibers and cancer cells, showcasing its potential value in cancer research.

Conclusions and Significance

The HILOTRUST technology, by combining HILO microscopy with UV excitation, achieves high-resolution, full-color 3D imaging with significant advantages in time efficiency and cost-effectiveness. This technique shows great potential in 3D histology applications, particularly in cancer diagnosis and embryonic development research. In the future, the research team plans to further optimize system performance, explore more types of fluorescent dyes, and expand clinical applications.

Research Highlights

1. High-Resolution and Full-Color Imaging: HILOTRUST achieves high-resolution, full-color 3D imaging through HILO microscopy and UV excitation, significantly improving image contrast and detail recognition.
2. Time Efficiency and Cost-Effectiveness: Compared to traditional 3D imaging technologies, HILOTRUST significantly reduces imaging time and cost while maintaining high resolution.
3. Potential for Clinical Applications: The imaging results of human lung cancer samples demonstrate the potential value of HILOTRUST in cancer diagnosis and research.

Future Prospects

Although HILOTRUST has made significant progress, there is still room for improvement. For example, future work could explore more types of fluorescent dyes to enrich imaging capabilities. Additionally, the introduction of deep learning techniques could further enhance imaging speed and image quality. As the technology continues to be optimized, HILOTRUST is expected to play a greater role in biomedical research and clinical diagnostics.