Featured Article
Monday December 07, 2009
by Caitlin Smith
Let’s face it: cells simply didn’t evolve to live in isolation, or even in monolayers, in little plastic dishes. How relevant are our experiments on primary cells or cultured cell lines? While we are far from answering this question completely, we do know that the technologies for replicating a cell’s environment in culture are evolving along with our instruments for measuring the processes that fascinate us. Here are some recent advances in the technologies used today for imaging live cells.
Time-lapse improvements
Among the most difficult types of live-cell experiments are time-lapse studies, in which changes in cells are observed over a longer time span. Cathy Owen, CEO and president of Nanopoint, says that stem cell differentiation is an important example: “Stem cells have the capacity to self-renew by cell division or differentiate into mature, specialized cells. During differentiation, certain genes get activated and other genes become inactivated in an intricately complex process. Differentiation takes place over an extended period of time, demanding technology solutions that utilize limited amounts of material and can keep the stem cells alive for multi-day experimentation.”
Nanopoint’s cellTRAY® Imaging System Model CT-2000 lets you run time-lapse studies with live cells over several days. With an integrated incubator and fluidics system for inverted microscopes, its cellTRAY holds a matrix of cells divided into six regions that can be separately addressed by the fluidics system for different reagent treatments. The cellTRAY also allows automated processing, and simultaneous monitoring of the matrix of cells. “Time-lapse imaging offers increased timing precision and repeatability, conserves on reagents, automatically captures events that can easily be missed, introduces the possibility of observing secondary results which may occur after the initial point of interest, while reducing the overall chance of user error,” says Owen. “Researchers can truly walk away while the system completely automates acquisition of the data.”
Olympus ’ integrated systems are designed to make live-cell imaging easier for all, even the novice. Their incubator microscope, the VivaView, can handle eight separate cultures for interrogation. “This allows the appropriate biological controls to be imaged under the exact same conditions of temperature, illumination, and camera exposure,” says Angela Goodacre, manager of confocal application development in the Life Sciences Group at Olympus America. “The optics are kept in thermal stability so that there is no drift in focus over long time-lapse experiments lasting over an entire week. We also have a confocal laser scanning microscope that sits on the bench, with its own darkroom, and built-in incubation. All you have to do is put the specimen in; it finds the focal plane and then you simply follow the prompts on the screen. The scanning parameters are optimized so you get the absolute best image quality, even if it's your first time on a microscope.”
Molecular Devices (MDS Analytical Technologies)’ ImageXpress Micro also offers live-cell capabilities for multiple-day time-lapse experiments with fluidics options. “Our XY stage is uniquely capable of excellent time-lapse movies with no jitter over the very long time courses that are often required, making it much easier to analyze cellular changes over time,” says Michael Sjaastad, director of marketing for imaging at Molecular Devices. “Imaging to monitor stem cell growth and differentiation both in stained and unstained assays” is an exciting new development in the field, he says. “The steady increase in use of live cell markers and fluorescent proteins in cells continues to amaze. This will greatly enhance our understanding of stem cell differentiation and lineage.”
To prevent movement of the cell plates during time-lapse experiments, BD Biosciences secures the plates themselves. “Our BD Pathway™ uses a fixed stage with movable optics, so there is no movement of the specimen over the course of the experiment,” says Khuong Truong, European sales and marketing manager at BD Biosciences. The BD Pathway™, a high-content cell analyzer for imaging, captures images in either confocal or widefield modes for best image quality. It also has both laser-based autofocus for rapid image acquisition, and camera-based (or combined) autofocus for more control during acquisition. The Pathway also allows follow-up of the growth and differentiation of individual cells over time, which Truong predicts will be of increasing interest in the near future.
Keeping cells healthy
Of course, cells didn’t evolve to be flashed with light and treated with dyes or fluorophores. “One of the most significant challenges for performing successful live-cell imaging experiments is to maintain the cells in a healthy state and functioning normally on the microscope stage for an extended period of time, while being illuminated in the presence of the fluorescent tags,” says Owen. “[For multiple-day experiments] just keeping the cells alive is a challenge that many live-cell imaging workstations cannot meet, and maintaining cells in an optimum physiological condition throughout the long observation phase is critical to the success of those experiments.” Nanopoint achieves this with environmental controls on the microscope, such as a miniature incubator to regulate temperature, humidity, and CO2 levels. Their Life Support Manager software controls the heater and syringe pumps for temperature and fluidics regulation.
A further challenge in maintaining culture conditions is trying to approximate the cells’ in vivo environment. “The biggest challenges in live-cell imaging lie in how closely the experimental system represents real biology,” says Goodacre. “[Growing primary or stem cell cultures] entails providing cells in vitrowith some semblance of the microenvironment they would have in vivo. Coatings such as basement membrane extract, three-dimensional matrices, and, recently, hydrogels, are increasingly used. Sometimes this means that imaging techniques that were developed for monolayer cultures need to be updated to take into account the three-dimensional nature of the cultures. We can help there, by providing optical sectioning methods and objective lenses that allow you to image deep into a culture.”
‘Next-generation’ imaging
One of the next techniques to come of age is label-free live-cell recordings, according to Khuong. “People are still working on it from an instrument and wet-lab standpoint, and it is probably healthier for the cells not to add label,” he says. “The problem right now, though, is that label-free techniques aren’t as sensitive as fluorophores for distinguishing between cells.” Goodacre agrees that “there are some very exciting developments that are making their way out of the physics laboratories and into the hands of biologists. The field of non-linear optics is coming up with some ways to image biomedically important molecules without even needing to add a dye or fluorescent protein. So, what you see is unperturbed by chemistry or genetic manipulation. Multimodal imaging, including CARS imaging of lipids, second harmonic imaging of collagen, and two-photon excitation of the intrinsic fluorescence of molecules like elastin—all at the same time—is going to be a tremendous tool for imaging live cells, and particularly live animals.”
Camera technology is advancing, too. Colin Coates, product manager for imaging at Andor Technology, says that they are developing a detector technology called Scientific CMOS (sCMOS). “The sensor we are launching with is 5.5 megapixel, yet is capable of running at 100 frames/sec with only three electrons read noise,” says Coates. “Slow down to 30 frames/sec and the noise floor drops to less than two electrons. This level of noise performance is attainable by only the very best CCDs under extremely slow readout. The pixels are 6.5 um in order to provide good oversampling of optical microscopy diffraction limits, yet the true dynamic range is an extremely impressive 20,000:1.” This is a leap from the best CCDs used for live-cell imaging today, which are capable of approximately 6 electrons read noise at 11 frames/sec, with 3000:1 dynamic range. Coates notes that “Andor’s sCMOS cameras will also be unique compared to previous CMOS technologies, in terms of offering less than 1% non-linearity and negligible fixed pattern noise—factors that have previously impeded the acceptance of CMOS for scientific usage.”
According to Coates, “the next big wave of advancement in live-cell imaging comes from super-resolution ‘nanoscopy,’ which offers the potential to specifically probe sub-cellular structural interactions at resolutions well below the classical diffractions limits. The biggest potential sub-diffraction improvements come from the stochastic photo-activatable single molecule techniques (PALM/STORM), which offer 20-30 nm in the axial plane and even 60-70 nm in the axial plane. There is a lot of proving to be done by these new systems with real living samples, but there is no doubt that within a few years the major hurdles of optical drift stability, data collection speed, and faster ‘real time’ processing will have been largely overcome.”