Live-Cell Imaging Tools to Visualize Organoids

Live-cell imaging makes it possible to study living cells, including organoids, in their natural environments over time. Organoids are three-dimensional microtissues grown in vitro from stem cells. In a controlled environment, the pluripotent cells mimic the structure and functionality of organs in vivo, making organoids vital tools for studying human physiology, disease biology, drug interactions, and much more. This article delves into the recent advances in live-cell imaging, along with industry best practices and tips for visualization success.

Recent advances in live-cell imaging technology

Live-cell imaging utilizes time-lapse microscopy to study dynamic processes occurring within living cells, in real time. “Multi-modal imaging techniques like fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) offer insights into the dynamic processes occurring within organoids, such as cell signaling and interaction,” explains Abdullah Ahmed, Ph.D., Global Business Excellence Manager, Life Science division at Leica Microsystems. “In addition, coherent Raman scattering allows users to image and differentiate structures and events using their chemical properties. This way, it can provide access to a vast amount of biochemical, metabolic, and pharmacokinetic information that is inaccessible via traditional methods. Such techniques can be combined in one with confocal fluorescence imaging.”

A few challenges that come with time-lapse imaging include phototoxicity and photobleaching or damaging the structural integrity of fragile organoids. “Innovations such as confocal technology with water immersion objectives help improve 3D imaging quality,” notes Amanda Jones, Senior Strategy Leader, Life Science Research, Revvity. “Also, the combination of effective dye combinations and automated high-throughput imaging is allowing for efficient analysis of 3D structures and experiments to be more easily scaled.”

Tips on minimizing or eliminating phototoxicity

• Utilize low-intensity illumination by using the lowest possible light intensity that still provides clear images to reduce cellular stress and damage.
• By optimizing imaging protocols to limit the duration and frequency of light exposure, researchers can further reduce exposure time. Furthermore, near-infrared fluorophores are less damaging to cells compared to visible light fluorophores.
• Advanced imaging techniques, such as light sheet fluorescence microscopy offers gentle, low-phototoxicity imaging, ideal for long-term studies.
• Multiphoton microscopy provides deep tissue imaging with minimal photodamage, suitable for thick organoid samples.

Tips provided by Valeria Chichagova, Director of Technology, Newcells Biotech

Organoid models have accelerated the development of drug screening methodologies. According to Valeria Chichagova, Director of Technology, Newcells Biotech, “High-content imaging platforms combine automated microscopy with image analysis software, enabling large-scale, high-throughput screening of organoids for drug discovery and genetic studies. They enable multiparametric readouts with more detailed information on the target of interest. Advanced algorithms for image analysis enhance the ability to extract quantitative data from complex organoid structures, improving the accuracy of phenotypic assessments. Techniques such as content-aware image restoration improve image quality and resolution from lower quality raw data.”

How dynamic imaging advancements are contributing to our understanding of organoid development and function

In live-cell imaging, real-time monitoring continuously monitors and records cells as they undergo various changes over time. A time-lapse sequence shows the progression of cellular behaviors and interactions such as migration, changes in cell shape, division, etc.

“Real-time imaging allows researchers to observe and track dynamic processes within organoids, such as cell proliferation, migration, differentiation, and morphogenesis,” explains Peter Favreau, Ph.D., Lattice Line Product Marketing Manager, ZEISS Research Microscopy Solutions. “By capturing images over time, researchers can analyze how cells within organoids respond to stimuli and interact with neighboring cells. Continuous imaging over extended periods also enables longitudinal studies of organoid development and maturation. Researchers can track individual cells or cell lineages over time, revealing heterogeneity in cell behavior and fate decisions. The advancement in dynamic imaging further aids in improving the accuracy and relevance of organoid models for studying development, disease modeling, drug screening, and personalized medicine.”

Non-invasive imaging methods

Minimizing physical damage to organoids, reducing the variability in data collection, and maintaining consistent experimental conditions are some of the advantages of non-invasive imaging techniques. “Excitation and illumination radiation needs to be minimized. This requires a photon efficient microscope with a short and simple optics path. Virtual staining is seeing success under the right circumstances,” explains Chris Shumate, Chief Executive Officer at Etaluma.

Shumate’s tips for success include:

• Placing the microscope inside the incubator. This ensures optimized growth environments for organoids.
• Labware motion acceleration profiles need to be reduced from those used on fixed samples.
• Tiling images over a large area reduces the need to physically locate the growing cells.
• Acquiring z-stacks and using z-projections and deconvolution gives 3D information that is easier to analyze.

In some scenarios, label-free imaging methods make it possible to study live cells without the need for fluorescent labels. Dr. Ahmed explains, “Label-free imaging methods, such as phase contrast microscopy, differential interference contrast (DIC) microscopy, or coherent anti-Stokes Raman scattering (CARS) microscopy, provide information about organoid structure and dynamics without the need for exogenous dyes or genetic modifications.”

AI advancements

AI continues to accelerate and influence development in the biotechnology space. "AI-driven methods, particularly deep learning algorithms, have demonstrated remarkable accuracy to segment cellular structures within brightfield images," Jones says. "One of the primary benefits is the ability to handle the subtle variations in contrast and texture that are characteristic of brightfield imaging, which traditional methods may not capture as effectively.”

Shumate adds, “Image analysis is changing fast. Meta’s ‘Segment Anything’ can allow voice commands to find and analyze objects in images. Image mode AI models will make analysis easy for anyone.”

Favreau notes that ZEISS ZEN Software provides comprehensive tools for image acquisition, processing, and analysis—“It supports advanced techniques such as machine learning and artificial intelligence to analyze complex imaging data from organoids. The ZEISS arivis Pro package can further enhance the image analysis capabilities, especially when dealing with larger 3D datasets.”

The convergence of artificial intelligence, advanced optical systems, data analytics, and creative use of illumination solutions, continues to facilitate the evolution of live-cell imaging.