Introduction
Live-cell imaging techniques are emerging as an invaluable tool for drug development, offering unprecedented insights into cellular behaviors and drug responses in real-time. This technology is particularly vital in cancer research, where understanding the dynamic interactions between drugs and biological targets yield profoundly more insight compared to static readouts. Combining live-cell imaging with patient-derived tumor organoid (PDTO) models could lead to even greater breakthroughs in therapy development. Here, we aim to compare two predominant live-cell imaging techniques – widefield and confocal imaging – and highlight their applications in the screening of PDTOs and more complex models such as assembloids and patient-derived tumor fragments.
Widefield Imaging vs Confocal Imaging
Widefield imaging is a foundational technique in microscopy that illuminates and captures the entirety of a biological sample in one planar field. This method captures images quickly, making it ideal for observing dynamic processes of organoids. In contrast, confocal imaging is a much more sophisticated technique that acquires high-resolution images by focusing on a single plane within a specimen. While this method is generally more time-consuming, the superior image quality acquired from confocal imaging allows for detailed insight into cellular and subcellular structures.
Advantages of Widefield Imaging for PDTO Screening
True to its name, widefield imaging is particularly effective for viewing the entire structure of a PDTO. This provides a high-level view of PDTO responses to various stimuli, making it a valuable tool in drug screening applications. Additionally, acquiring widefield images is faster and cheaper than confocal imaging and requires less storage capacity. Therefore, widefield imaging is an excellent tool for capturing functional organoid response (e.g. cytostatic vs cytotoxic) to therapeutics, especially in higher throughput applications.

Another major advantage of widefield imaging is the potential for label-free image analysis using AI image segmentation techniques such as Orbits, either from brightfield or phase-contrast images. Fluorescent labels, used to track cells, cellular structures and biological processes during live-cell imaging, are known to affect basal cellular activity via inducing oxidative stress through the process of photoxicity. Therefore, these reagents can skew experimental results and confound the effects of drugs in a screening. Whereas confocal requires these labels, widefield imaging can eliminate the need for fluorescent labels, giving researchers a non-invasive method to observe biological responses and avoid confounding effects on therapy responses.
Advantages of Confocal Imaging for PDTO Screening
Confocal imaging produces a much sharper and detailed image in one plane of focus. Furthermore, images from each plane of focus can be stacked to reconstruct a 3D structure. The major advantage of this method for detailed studies of the cellular and subcellular structures. However, confocal imaging often requires more technical handling, longer acquisition times, and more expensive equipment.

In the context of PDTOs, confocal imaging excels in analyzing single cells within tumor organoid structure. The ability to produce clear images of individual cells within a complex 3D structure is invaluable in understanding tumor heterogeneity and delineating interactions, particularly in co-culture systems that can include tumor cells, immune cells, fibroblasts and endothelial cells such as assembloids and tumor fragments. Currently, confocal imaging is bound to the use of fluorescent reagents, but with the rise of computer vision and AI technology, label-free PDTO analysis may become commonplace in the future. It's important to note that there is a risk of phototoxicity associated with the use of these fluorescent reagents, which might affect therapy response, especially for therapies that affect the cellular redox systems.
Conclusion
Widefield and confocal live-cell imaging techniques each have unique strengths and limitations in the context of tumor organoid screening. While widefield imaging offers a broader view of PDTOs, making it suitable for general drug screening, confocal imaging provides detailed insights at the cellular level, critical for understanding complex tumor microenvironments and cell-type specific responses. The choice between these techniques will always depend on the specific requirements of the research project. By understanding these differences and applying the appropriate method, researchers can gain deeper insights into tumor biology and therapy responses, paving the way for more effective cancer treatments.
Our Expertise
For high-throughput PDTO screening, widefield imaging stands out as the optimal choice. Our expertise in this field is underscored by the capabilities of our proprietary image and data analysis software, Orbits. With Orbits, we can extract a wealth of critical information from brightfield images at the single organoid level, achieving a remarkable correlation with clinical responses. This fully automated approach not only streamlines the screening process but also ensures that the insights gained are highly relevant to real-world outcomes. However, we understand that cancer research often necessitates more complex models that encompass various cell types within the tumor microenvironment (assembloids and tumor fragments). To address this need, we provide advanced confocal imaging solutions. Confocal imaging enables us to delve deep into the intricacies of cell-type-specific therapy responses and cellular interactions within these multifaceted tumor ecosystems. Our commitment to versatility and precision allows us to support researchers in gaining comprehensive insights into tumor biology and therapy responses, ultimately driving advancements in the field of cancer treatment.
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