Sometimes, technology drives research in the life sciences. That has happened with live-cell imaging, because sophisticated platforms are necessary to make this technology possible. However, with live-cell imaging, what really matters are what scientists can see and the implications for their research.
In some ways, this technology comes from one of the oldest pages in the playbook, actually the baseball one. “As Yogi Berra said, you can observe a lot by just watching,” says Jeremy Baskin, Nancy and Peter Meinig Family Investigator in the Life Sciences at Cornell University. In the biology of cells, there’s lots to watch.
“The main benefit of using live-cell imaging is the observation of dynamic events in cells and the observation of ‘moving’ biology rather than fixed cell biology,” says Scott Olenych, North American product marketing group manager, light microscopy at Carl Zeiss Microscopy. “Secondary benefits include seeing the interactions of proteins and molecules in real time.”
The broad benefits of this technology push it increasingly into mainstream biology. As Karin Boettcher, associate product manager for high-content screening and applications at Revvity, says, “Live-cell imaging is becoming a requisite technique for cell biology, developmental biology, cancer biology and many other related biomedical research laboratories, and it has gained importance within drug discovery over recent times, as researchers look for more meaningful insights into cellular behavior and function.”
As time goes on, even more benefits of this technology will surely emerge.
Why go live?
Even today, this technology can be used to address a range of biological mechanisms. As examples, Werner Wittke, head of application management for widefield microscopy at Leica Microsystems, mentions that live-cell imaging can be used to study cell-cell interactions, understand intracellular machinery, influence and image fluorescent proteins inside living cells, and more.
Different biologists use live-cell imaging for their own purposes, but some general themes can be found. When asked about the top-three benefits of using live-cell imaging, Will Marshall, product manager for high-content analysis at GE Healthcare Life Sciences, picks out: “biological relevance, a nice way to answer open-ended questions and temporal changes provide tons of information.”
For biological relevance, Marshall says, “Anyone who has done imaging is well aware that there is potential for inducing artefacts when fixing cells, using hardening mounting media, etc.” Those are some of the things that live-cell imaging avoids, thereby making imaging more valuable, but there’s more. “Many experimental readouts are more informative when looking at dynamic measurements and behavior,” Marshall explains. Although he points out that some tweaks in an assay allow a biologist to study cell-cell interactions in fixed cells, “actually watching the cells interact and understanding what they were doing before and after interaction can add another layer of information that may be more representative of what is important in vivo.”
Sometimes, the most intrigue arises from open-ended questions. “In previous work in the lab, researchers often asked me to help them understand something very high level like: describe how the cyst forms,” Marshall recalls. “With lots of supporting information, you could design an experiment that focuses on a specific piece of cyst formation, perform a few more of these experiments and then piece back together the big picture.” But, that was the hard way. Live-cell imaging over time, though, could provide the answer in one experiment. “After seeing the event in its entirety, you can always follow up with very specific analyses and work backward to quantify specific parts of a complex process,” Marshall says.
The cyst example suggests some of live-cell imaging’s temporal benefits, but it can go further. “I’ve seen groups take this to another level with some real data mining,” Marshall explains. “For example, clustering compounds by the rate at which they induce a phenotypic change could give you an insight into the mechanism of action of an unknown compound.”
Benefits of balance
In many ways, live-cell imaging arises from scientific interest coupled with improvements in labeling and imaging technology. “There have been exciting advances in the area of probe development that enable a wider array of proteins, nucleic acids, glycans, lipids, metabolites, ions, and other targets to be labeled,” Baskin explains. “Separately, advances in microscopy—in particular, various techniques that circumvent the diffraction limit and still allow for rapid image acquisition with minimal phototoxicity to cells—allow the experimenter to visualize these molecules in live cellular contexts with, in some cases, near-molecular resolution.”
Putting together the technological advances with the biological interest gives scientists even more ways to use live-cell imaging. “These parallel advances in probe development and microscopy methods—many of which are compatible with not only live cells but live organisms—allow observations to both generate and test mechanistic hypotheses,” Baskin states.
Image: In this live HeLa cell, fluorophore-tagged lipids reveal phospholipase signaling activity (green), and a fluorescent protein-tagged marker labels the endoplasmic reticulum (magenta). Image courtesy of Timothy Bumpus, a graduate student in Jeremy Baskin’s lab at Cornell University.
Learning even more from living cells requires imaging techniques that cause less damage. As an example of that, Olenych mentions the Lattice Light Sheet technology developed by Eric Betzig and his collaborators at Janelia Research Campus in Virginia. “Lattice light-sheet allows extremely gentle imaging of live cells with high speeds and very low or no cell damage,” Olenych explains.
Going 3D
Other advances will also expand the applications of live-cell imaging. “Recent advances in microscope technology and software are enabling a better quantitative image analysis of label-free images,” Boettcher notes. “By measuring the refractive index of the sample and how that changes in 3D and over time, this technology enables color-coding images or creating a digital staining that provides structural and chemical information about the cell’s interior without adding any dye or genetic modification.”
Some of the deepest mysteries of all might even be addressed through live-cell imaging. “With new advances in modelling approaches, I really am interested in any work that monitors neurons firing and communicating across large distances in a brain organoid,” Marshall says. “In combining brain-like organoids and live-cell imaging, Marshall envisions eventually “mapping such a complex network of interconnected signaling events.”
Wittke agrees on the value of imaging live organoids. He says that these structures can be used to “image and understand cells in their ‘natural environment’ as a tissue—in this context, the generation of artificial tissue.”
Studying cells in a more realistic environment triggered this field from the start. Ongoing advances are driving applications of live-cell imaging that get increasingly close to watching cells as they exist and operate in nature.