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3D biology

Recent advancements in life sciences have prominently featured the development of 3D cell biology. This includes the use of multicellular structures like tissue sections and the creation of 3D culture systems such as spheroids, organoids, and organ-on-a-chip (Microphysiological system). These innovations mark a significant departure from 2D cell cultures, which, while valuable, are inadequate in accurately mimicking the complex spatial cellular organization and tissue dynamics found in vivo.

In 3D biology research, conventional imaging techniques have inherent limitations. The use of exogenous agents for labeling or staining often interferes with cellular functions and compromises data reliability. Additionally, it remains challenges to capture the complete axial structures of thick specimens, such as organoids, or to accurately depict the tissue microenvironment, including dynamic fluid flow, mechanical cues, and tissue-tissue interfaces.

Tomocube Holotomography (HT) is ideally suited for research utilizing 3D cell cultures. The HT-X1 imaging platform offers several key advantages: (1) the ability to capture the complexity of 3D structures, (2) accessibility for non-invasive, live observation of tissue dynamics, and (3) quantitative measurement capabilities suitable for high-throughput applications.

First, HT visualize biological samples by capturing its structural information through light wave, encoding this information onto 2D intensity images, then reconstructing these images into a 3D tomogram for three-dimensional visualization. Through optical sectioning, the HT-X1 enables visualizing depth-specific slices of thick samples and 3D image. Its fast scanning speed and absence of speckle noise are remarkable features in 3D biology applications, ensuring a high-contrast view of the dynamic complexity within 3D models.

Second, HT utilizes refractive index (RI) as image contrast. By minimizing the need for labeling, it preserves cell viability and enables long-term monitoring of cellular dynamics without the risk of photobleaching or phototoxicity. Imaging with HT-X1 allows 3D samples to be observed in their intact forms and without time-consuming, labor-intensive sample preparation protocols. This preserves data reliability and reducing burdens on researchers. Additionally, its fast acquisition speed provides high temporal resolution, capturing rapid cellular events.

Furthermore, HT facilitates quantitative measurement of 3D tissues and organoids, including size, volume, mass, and growth rate. This capability supports comprehensive, longitudinal tracking of the samples' characteristics and can be leveraged in high-throughput efforts for assessment of drug response.

In summary, Holotomography addresses the limitations of existing imaging techniques by providing high-resolution, label-free imaging of 3D cell cultures. Its noninvasiveness, combined with fast scanning speeds, high-contrast visualization, and quantitative measurements, makes HT an invaluable live cell imaging tool for 3D cell biology.

Features

  • Larger Field-of-View
    Label-free live cell imaging
    Holotomography (HT) imaging eliminates the need for labeling or dyeing, therefore minimizing artificial manipulation effects and simplifying sample preparation. This approach allows the observation of samples in their natural state, providing more reliable and relevant information.
  • Larger Field-of-View
    High-resolution 3D imaging
    HT provides high-content, high-resolution, three-dimensional images, allowing researchers to capture detailed information about cellular structures. This high level of detail is crucial for studying cellular morphology and dynamics.
  • Larger Field-of-View
    Long-term timelapse imaging
    HT facilitates continuous monitoring of cellular phenomena over extended time frames. This feature is beneficial for studying processes that unfold over hours, days, or even weeks, providing a comprehensive understanding of long-term cellular behavior.
  • Larger Field-of-View
    Quantitative analysis
    HT allows for quantitative analysis of cellular parameters. It can measure physical quantities such as mean refractive index,dry mass, and concentration, as well as various shape measurements like area,length, volume, and surface area.

Discover 3D biology with HT

  • Organoids

    Research on organoids, the 3D cell aggregates replicating organ structures and functions, benefits significantly from the detailed and noninvasive imaging capabilities of Holotomography (HT). HT efficiently captures the dynamic behavior of cells within living organoids, including mitotic events, cyst formation, and crypt budding. Real-time, high-resolution observations with available quantitative measurements facilitated by HT are crucial for exploring the complex dynamics and morphology of the organoids.

    In combination with the HT-ready 96-well plate, which supports a broad spectrum of cell types from 2D to 3D cultures, the HT-X1 is an ideal tool for leveraging the potential of organoids in drug development. It enables high-throughput screenings for rapid assessment of drug response and its toxicity on patient-derived tissues.

    A recent study highlighted a successful example of such efforts, demonstrating how HT has set a new standard for comprehensive and rigorous statistical assessments for the biological studies of organoids (Lee et al., 2023). With HT, intricate subcellular structures of individual cells in small intestinal organoids were observed in intricate detail. Long-term monitoring of various biological processes within the specimen, including cell mitosis, migration, apoptosis, and other subcellular dynamics, was achieved effortlessly without the need for labeling or other time-consuming preparations.

    Additionally, the observation of morphological changes was combined with quantitative parameters, including organoid volume, protein concentration, and mass, to effectively and comprehensively evaluate the response of the organoids to chemotherapy drug treatments. The study suggested that the Tomocube HT-X1 imaging system is an indispensable tool for organoid-based pharmacological screening applications.

  • Advanced scaffold-based 3D cell cultures – Organ-on-a-chip

    While 2D cell culture systems have been instrumental in advancing our understanding of cell biology, they fall short in replicating the natural microenvironment of tissues due to the lack of cellular communication and interaction. Therefore, it is essential to study cell and tissue behavior in a 3D context that mirrors the structure of the extracellular matrix (ECM).

    Among various 3D cell culture techniques, organ-on-a-chip is in increasing demand across diverse biomedical research fields. Unlike less advanced 3D culture systems, such as protein-based ECM or hydrogels, organ-on-a-chip combines cell culture with microfluidic devices (µFDs). By simplifying and miniaturizing integrated biological processes on microscale chips, it better imitates the dynamic mechanical properties and biochemical functionalities of living organs.

    The HT-X1 is a powerful tool that offers numerous benefits for organ-on-a-chip research. It provides a precise representation of cellular behavior in a 3D environment with high resolution and in real-time, enabling researchers to better understand cell interactions in complex environments. Additionally, the HT-X1 can track cellular behaviors without relying on staining techniques, which is particularly useful when fluorescent staining is challenging in intricate 3D platforms.

    The excellent ability of Tomocube low-coherence HT was demonstrated in an experiment observing live Hep3B cells on a 3D collagen type I and fiber structure within the DAX-1 chip (video). High-resolution images of live Hep3B cells were obtained, featuring detailed structures of organelles such as the nucleus, nucleolus, lipid droplets, mitochondria, and collagen fibers at exceptional spatial resolution. The Hep3B cells exhibited a distinct round-shape morphology that differs from their morphology in a 2D environment, highlighting the significant influence of the ECM on cell behavior and morphology.

  • Tissue

    Study tissues provides a more comprehensive and accurate understanding of biological processes, disease mechanisms, and therapeutic responses compared to studying isolated cells. However, research involving tissues presents numerous challenges. These include technical difficulties such as maintaining tissue integrity, manipulation, and culture, as well as technological limitations in imaging and analyzing tissue heterogeneity and structural complexity.

    Holotomography (HT) is a pivotal advancement in live tissue imaging. It optimizes tissue research by offering deep structural insight of intact tissue sample without the need for arduous preparation steps such as thin tissue sectioning, fixation, and staining, which often compromise the morphological and physiological information obtainable from the specimen.

    A recent review highlights the inevitable transition from 2D to 3D spatial profiling in cancer research, showcasing how HT has transformed our understanding of cancer cells and their surrounding tumor microenvironments (Wang et al., 2024). Tomocube’s 3D imaging technology addresses challenge such as extensive sample preparation, preservation of tissue's natural state, and post-imaging multimodal molecular data collection. These innovations are paving the way for the future of personalized medicine, enabling more precise and effective cancer diagnosis and treatment.

    The video on the right showcases HT imaging capabilities, revealing remarkable spatial features of three different mouse reproductive tissues. Observations included the dynamics of live placental tissue, stages of spermatogenesis within seminiferous tubules, and detailed structures in oviduct tissue. Using HT, the morphology of epithelial cells, sperm cells, cilia cells, secretory cells, and the lamina propria were all visualized in high detail and in 3D. The timelapse capture of internal flow and beating in placental tissue offers additional insights into reproductive processes.

Resources

Selected publications

  • Research team proposed a new lung biopsy technique - cryobiopsy - resulting in a higher success rate for culturing lung cancer organoids (LCOs) while overcoming many critical limitations of conventional organoid models. Low-coherence HT was employed to evaluate the 3D structure and subcellular characteristics of LCOs without any pre-treatment. This proposed methodology promises to be a breakthrough strategy for the clinical application of LCOs in all stages of lung cancer.

  • HT sets a new standard for comprehensive, rigorous statistical assessments for the studies of organoids. With HT, researchers can observe intricate subcellular structures of individual cells in small intestinal organoids and easily perform long-term monitoring of different biological processes as the procedures required no time-consuming preparation or labeling. Observation of morphological changes was combined with quantitative parameters to effectively and comprehensively evaluate the response of the organoids to drug treatment.