Live-cell imaging provides opportunities for understanding cell physiology that cannot be discovered by other methods. It is especially important for studying processes that occur over longer time scales, such as cell division, development, and migration. A central challenge during live-cell experiments is maintaining the cells in conditions that encourage them to thrive—otherwise, results might be due to unsatisfactory conditions rather than intentional experimental manipulation. While researchers sometimes face a trade-off between cell health and image quality, it is true that healthier cells yield higher-quality data. This article offers advice from experts on optimizing cell health and live-cell imaging.
Healthy cell cultures
Monitoring cell health is critical for carefully controlling culture conditions. Keeping cultured cells in peak condition requires nonfluctuating levels of many important variables that impact cell health: temperature, media pH and osmolarity, cell confluence, oxygen level, carbon dioxide level, and humidity. “Standardizing this portion of your workstream with objective, image-based analysis minimizes the impact of these variables, which otherwise could adversely affect the results and data obtained from downstream assays, ultimately wasting valuable time and money,” notes Belinda O’Clair, Head of BioAnalytics Applications, Reagents and Consumables Product Management at Sartorius.
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A live-cell imaging system entirely housed within an incubator is a convenient solution, as long as the size is right. “One should [ensure] that there is enough space around the [imaging system] in order to maintain a proper airflow inside the incubator,” says Inge Thijssen, Senior Application Scientist at CytoSMART. “If the live-cell imaging device fills up the entire incubator, this might lead to issues in maintenance of the proper environmental conditions.”
In addition to enclosed or microscope stage-top incubators, other tips to keep cells healthy include heating the microscope objective, and eliminating drafts to help stabilize the room temperature. With multiwell plates, using only the inside wells can also help to stabilize temperatures, which are more likely to fluctuate in wells along the edges of the plates.
Make every photon count
Another priority is minimizing phototoxicity and photobleaching by using the lowest light intensity and the shortest exposure times,possible to obtain results. “It is important to make every photon count by keeping unnecessary excitation to a minimum and capturing the maximum of the emitted photons,” says Karin Boettcher, Strategy Leader for Biotech Research in Life Sciences at Revvity. Optimizing the light path to capture more emitted light, and using a more sensitive photo detector, can help to reduce light exposure times. Designing experiments to minimize illumination time is another strategy to protect cells from excess light.
Potential pitfalls
Don’t assume validation of reagents
Continuous live-cell imaging assays might require different reagents than endpoint assays. “Researchers often infer that reagents used and validated in end-point experiments translate for use in continuous live-cell experiments, which simply is untrue,” says O’Clair. These reagents may contain high concentrations of DMSO or additives such as sodium azide, which impact cell health.
Pay attention to cell seeding
In multi-well plates, non-homogeneous cell seeding and edge effects can be sources of unwanted variability. “The causes of non-homogeneous cell seeding can be attributed to thermodynamics within a well and can cause well-to-well variability,” says O’Clair. “In order to avoid this, we recommend incubating newly seeded plates at room temperature before placing them in an incubator for imaging.” Advances in automated cell seeding have also been helpful.
Pay attention to your microscopy set-up
Even the microscope set-up itself can affect the cells’ environment. “Some live-cell imaging set-ups contain large motors to move the stage, which can lead to an increase in temperature every time the stage is moved,” notes Thijssen. “Therefore it is very important to use a system with low heat production or allow for sufficient time between imaging timepoints for the system to cool down.”
New technologies are helpful for optimization
More data at once
Today, imaging systems can detect and analyze more than ever. “Systems with the capacity to visualize entire samples provide revolutionary amounts of data,” says Thijssen. “[Some can] visualize all cells within large petri dishes or T-flasks, making them highly suitable for cell therapy labs.”
Brightfield microscopy
Brightfield microscopy can now detect many things that researchers used to rely on fluorescence imaging for, which can be helpful if fluorescence is causing too much photodamage. “Current image analysis algorithms have become increasingly better at detecting many cellular features using solely brightfield microscopy,” says Thijssen. “So scientists need to think carefully about whether fluorescence is absolutely necessary in their experiments.” Some live-cell imaging systems have integrated image analysis algorithms for brightfield imaging, but researchers can also use freely available image analysis tools.
Deep learning for lower light levels
New computational tools are emerging to help researchers analyze imaging data from modalities that typically incur less photodamage, such as brightfield, phase contrast, or differential interference contrast (DIC) microscopy. But segmenting these data—an important step in the process of microscopy image analysis—requires new and improved methods. Fluorescent imaging data, which is typically high contrast, is easier to segment.
“Segmentation of brightfield, label-free images is notoriously challenging due to the lack of contrast in the images and bright halos around the cells and artifacts,” says Boettcher. Computational approaches such as machine learning and deep learning, which make fewer segmentation errors compared to conventional imaging analysis, offer promise for future imaging modalities. “Such developments have the potential to broaden the scope of live-cell imaging assays and enable numerous experiments not possible before,” she adds.