Using common research techniques for characterizing organoids presents challenges that include low throughput and insufficient depth of information. Here, we look at some of the novel systems developed to address these limitations and share tips for successful 3D imaging of organoid cultures.

Limitations of common methods

Organoid culture has revolutionized scientific research by providing a means of modeling complex mammalian tissues in vitro. However, applying common research techniques for studying organoids is not always a viable option. “When organoid culture was first established, characterization typically involved paraffin-embedding and sectioning the samples before immunostaining for various tissue-specific and stem cell markers and visualizing the organoids with conventional microscopy,” reports Oksana Sirenko, Ph.D., Senior Scientist at Molecular Devices.

“In addition, transcriptomic analysis was used for monitoring organoid differentiation over time. While extremely informative, these methods were too slow and labor-intensive to be used for compound screening or drug development. As such, high-throughput analysis approaches for detecting discrete morphological and functional changes in organoids subsequently evolved. Now, to better capture the depth of information offered by organoid cultures, researchers are leveraging more advanced technologies such as automated high-content imaging platforms.”

Imaging challenges

Compared to 2D imaging of cell monolayers or tissue sections, 3D imaging of organoid cultures poses multiple challenges. “Firstly, the thickness and opacity of organoids can limit light penetration, making it difficult to capture detailed information from the inner layers with conventional microscopy techniques,” explains Joe Clayton, Ph.D., Global Scientific Program Manager at Agilent Technologies. “Secondly, the dynamic nature of organoids necessitates time-lapse imaging, which can lead to phototoxicity and photobleaching. Maintaining the structural integrity of fragile organoids during imaging is another concern, while the sheer volume of data generated from 3D imaging can quickly overwhelm data storage capacity and computational resources. Addressing these challenges often requires a combination of advanced microscopy techniques, specialized imaging equipment, and innovative data analysis methods to obtain meaningful information.”

Another complication stems from how to keep the biology behaving naturally during the imaging process. “Long-term growth of organoid cultures requires media exchanges, making on-deck incubation chambers problematic,” notes Chris Shumate, CEO of Etaluma. “One way of overcoming this issue is to place the entire microscope within the cell culture incubator, hypoxia chamber, or sterile glove box. This allows feeding and media exchanges within the culture environment, ensuring that physiological conditions are maintained throughout a multi-day or multi-week time-lapse experiment.” Shumate also cautions that some microplate labware that is optimized for organoid development is non-optimal for imaging. “Round-bottom and inverted pyramid shaped wells especially pose imaging challenges,” he says.

Recent advances

Many recent discoveries within organoid research can be attributed to advances in instrumentation. These include confocal technology and the combination of water immersion objectives with lasers. “Confocal systems often employ spinning disks with tiny holes that significantly minimize light scattering from out-of-focus regions during imaging,” says Sirenko. “While this approach results in reduced light intensity, as only a fraction of light passes through the holes, this is counteracted with the use of powerful light sources like lasers to enable high-resolution imaging. Water immersion is used to further enhance image quality, intensity, and resolution.”

In addition, automated imaging-based approaches within microplate formats deliver considerable improvements over conventional manual microscopy methods in terms of throughput, reproducibility, and robust, real-time analysis. Clayton notes that this strategy forms the basis of the recently released Agilent BioTek Cytation C10 Confocal Imaging Reader, which combines spinning disk confocal and widefield imaging with a multimode reader for increased application versatility. “Features of the Cytation C10 designed to enhance 3D imaging of organoid cultures include advanced imaging attributes, such as automated focusing methods and beacon (ROI) selection in x, y, and z dimensions for capturing specific areas of interest, and multichannel z-stack imaging combined with Gen5 software tools for intuitive viewing of entire organoid samples,” he says. “Additionally, the Cytation C10 can be integrated with the Agilent BioTek BioSpa Automated Incubator for long-term live cell imaging applications.”

Improvements to image analysis have also been fundamental in allowing researchers to delve deeper into organoid cultures. Advanced analysis software include Molecular Devices’ MetaXpress® High Content Image Acquisition & Analysis Software, which is capable of intricate measurements such as counting and characterizing cells within organoids, analyzing subcellular objects, and detecting proximity and clustering, and its AI-powered IN Carta® Image Analysis Software, which uses machine-learning tools to recognize different object types and distinguish diverse morphologies or features. “Importantly, these innovations unleash restrictions around the single-measurement approach to let researchers obtain the more complex information available from organoid cultures,” comments Sirenko.

At the same time that instrumentation and analysis software have become more powerful, various reagents have been developed to enhance 3D imaging of organoid cultures. These include products that turn tissues transparent, such as Corning® 3D Clear Tissue Clearing Reagent. “Clearing reagents reduce light scattering and improve light penetration by normalizing the refractive index throughout the tissue,” explains Hilary Sherman, Senior Applications Scientist at Corning Life Sciences. “Specifically, by matching the refractive index of the cytosolic and intercellular space of organoids to that of the cell membranes and proteins, Corning 3D Clear Tissue Clearing Reagent enhances the capabilities of an imaging system even further.”

Tips for successful imaging

There are multiple steps that researchers can take to ensure successful 3D imaging of organoid cultures. “Like with any imaging experiment, it’s important to optimize the protocol,” says Sherman. “Sometimes penetration of antibodies and stains can be more challenging with larger 3D structures like organoids, so testing different concentrations, permeabilization techniques, and incubation times is essential.”

“You may wish to consider using a growth medium or buffer designed for fluorescent imaging applications to reduce background or autofluorescence,” adds Clayton. “Also, remember to select fluorescent labels with excitation and emission profiles that are well-matched to the imaging system. If practical, the incorporation of genetically encoded fluorescent labels can provide more consistent labeling throughout the organoid.”

Sirenko highlights the value of targeted imaging protocols, which initially capture wells with low magnification before focusing on organoids for imaging with higher magnification. “For basic counting and growth observation, a lower magnification like 4X is recommended,” she says. “10X offers ample resolution for organoid analysis within the field of view. To reveal finer details, higher magnifications like 20X and 40X are needed, although these may require stitching different images to capture an entire object.”

Lastly, Shumate suggests that the biggest learning curve for researchers new to extended time-lapse microscopy is the trade-off between imaging interval and photo-effects. “Often researchers have little experience with their organoids under extended light exposure protocols,” he says. “At Etaluma, we recommend reserving a few wells of an experiment for only time = 0 and time = final images for comparison with those which were imaged repeatedly over the same period. Phototoxicity can vary widely between different cell types and the imaging interval as well as the gain/illumination settings may need optimizing based on these results.”

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