Recent developments in environmental control technology in live-cell imaging—maintaining constant temperature, CO2 levels, and reducing photodamage—are fueling further advances in the application of live-cell imaging to central questions of cell biology. Now that most live-cell imaging systems provide a better living environment for cells, researchers are developing tools to address the biological curios that drive their work, such as cell migration, signal transduction, and cell division. Here’s a look at some live-cell imaging systems that are allowing scientists to begin unraveling the mysteries of how cells self-regulate and thrive.
3D spheroid assays and cell monitoring
As 3D spheroid assays become more prevalent, systems that monitor cell health within incubators are increasingly valuable. Leica’s PAULA Smart Cell Imager is designed for routine cell culture monitoring, and includes alerts for desired confluency or transfection state (as do many others). Sartorius also offers a portfolio of IncuCyte Live-Cell Analysis systems for automated image acquisition and analysis of cell cultures within the environment of a tissue culture incubator.
Originally developed to monitor cell health and confluence of cultures, “the IncuCyte allows scientists to study dynamic biological changes while controlling variables that can adversely affect the results and interpretation of data,” says Belinda O’Clair, global product manager for IncuCyte at Essen BioScience (a Sartorius company). “Controlling these factors allows researchers to study long-term biological changes from robust cancer cells to extremely sensitive cells, such as human induced pluripotent stem cell-derived neurons.”
Essen BioScience’s IncuCyte system includes applications for 3D tumor spheroid assays. “We offer an integrated turnkey solution that began with understanding the cell model to be studied, and then used that knowledge to design acquisition and analysis tools to quantify tumor spheroid formation, growth, health, and invasion,” says O’Clair. A suite of applications for the IncuCyte also supports different therapeutic research areas, including “complex questions of quantitating spatial and temporal changes in such assays as antibody internalization, cell migration and invasion, as well as neuronal activity,” she says.
Etaluma’s automated LS720 microscope, and its accompanying analysis software, is well-suited for studying 3D spheroids because z-stacks of images can be acquired in user-defined positions. “Many times, the spheroid is not in the center of a pre-defined well, and so custom ‘region of interest’ positions can be defined prior to the imaging experiment,” says Chris Shumate, CEO and co-founder. Etaluma’s “Pixel Classifier” (in partnership with DRVision) feature allows users to denote objects in the image that should, and should not, be measured, which is helpful for users with little image analysis experience.
Time-lapse imaging
Also supporting 3D spheroid cultures are systems made by CytoSMART Technologies and Etaluma, which support long-term and time-lapse experiments. CytoSMART’s Lux2 is a compact inverted microscope, designed to image cells within any regular CO2 incubator. The recorded images are stored and analyzed in the CytoSMART Connect Cloud, which is accessible from any remote device for real-time monitoring. “The CytoSMART Lux2 can be used across a wide range of applications, including cell division imaging, cell growth and confluence monitoring, cell migration, wound healing and scratch assay analysis, stem cell behavior studies, and chemotaxis studies,” says Joffry Maltha, CEO of CytoSMART Technologies.
For high resolution, full well, live-cell imaging, the CytoSMART Omni can scan a 96-well plate within six minutes. “The integrated confluency analysis software of the CytoSMART Omni enables scientists to perform apoptosis, proliferation, cytotoxicity, migration, and colony formation experiments in just a few clicks,” adds Maltha.
Etaluma makes bright field, phase contrast, and epi-fluorescence inverted microscopes that operate within incubators, which is essential for time-lapse microscopy. “Our customers routinely perform multiday and even multiweek long imaging experiments,” says Shumate. In addition to monitoring cell health, their scopes are also used in studying 3D organoids and spheroids, monitoring lab-on-a-chip microfluidics, kinetic biosensors, cardiac myocyte beating measurements, and oocyte development studies. “Co-cultures are on the rise for CAR-T and other immuno-oncology applications,” Shumate adds.
Newer approaches
Ryan Hrejsa, senior marketing manager in life science research at Leica Microsystems, says that scientists routinely use Leica imaging solutions “to probe dynamic interactions between molecules, 3D cell culture, or cellular organization in organoids.” He believes that new approaches to live-cell imaging are on the horizon, for example, using optical sectioning of 3D cell cultures—the Leica SP8-DLS uses a digital light sheet method, while the Leica THUNDER Imager uses computational clearing. “[These] are perfectly suited for high-speed 3D imaging, with reduced light exposure to live cells and highly sensitive detectors,” says Hrejsa.
New imaging modalities are also coming to live-cell imaging, such as “FLIM imaging, available on the Leica SP8 FALCON, to probe the microenvironment inside live cells,” explains Hrejsa. FLIM (fluorescence lifetime imaging microscopy) looks at the lifetimes of fluorophores in the excited state, rather than their emission spectra, and can differentiate between two fluorophores of the same color. Hrejsa believes that another growing area is correlative electron microscopy (CLEM), in which live cells are first probed with optical imaging, and then fixed and correlated with electron microscopy imaging of the same sample. “The Leica Cryo CLEM solution can provide this link between live-cell imaging, and the molecular-level resolution obtained only on an electron microscopy platform,” says Hrejsa.
Future directions
Live-cell imaging is poised to advance in many directions. Maltha believes that kinetic (time-based) experiments will become increasingly significant, versus traditional end-point measurements. “Kinetic data can provide greater insights into cell behavior as a result of different stress factors,” he says. “Overall, kinetic data will enable scientists to study even minor and less frequent effects, those that occur in only one part of the cell population, and long-term effects.”
Shumate also predicts that optogenetics, a technique that uses light to trigger the activity of genetically defined cells, will become more important. “Optogenetics offers a stopwatch for initiating biology,” he says. “The ability to trigger biology while imaging is very powerful.”