Valerica Raicu, Ph.D., assistant professor, departments of physics and biological sciences, University of Wisconsin at Milwaukee, has built a new imaging instrument that provides full information on interactions and spatial distribution of protein complexes in live cells in a single scan.
“We designed and built a new instrument,” Dr. Raicu says, with the goal of commercializing it. In contrast to the two-photon microscope, which builds three-dimensional images from planar sections of a sample, this instrument takes a multidimensional image using the full spectrum of wavelengths. “You then can reconstruct the image at different wavelengths,” he adds.
In his work with GPCRs, his instrument revealed the relative disposition of molecules in the complex, showing four monomers, arranged as a parallelogram. “We started a new business: the structural determination of protein complexes at molecular resolution,” Dr. Raicu explains.
Next, Dr. Raicu’s lab plans to develop a faster version of the instrument to take a series of snapshots to reveal intracellular dynamics, focusing on protein complex trafficking and what happens to the structure after ligand binding.
At Childrens’ Hospital Boston, Thorsten Schlaeger, Ph.D., head hESc core facility, is using live-cell imaging to study the reprogramming process, in which fibroblasts are converted into induced pluripotent stem cells. “Prior studies generally relied on cell fixation, single markers, and single time point analyses that not only misidentified the true state and fate of the majority of cells, but also failed to reveal the complexity of reprogramming,” he says. Additionally, the process typically has much less than 1% efficiency and takes one to two weeks. “These are significant limitations to image-based analyses,” Dr. Schlaeger reports.
To overcome that, his lab used the BD Pathway Multi-Color Live Immunofluorescence Imaging platform (BD Biosciences) “to observe several square centimeters to allow for multiple bona fide reprogramming events to be captured over time,” Dr. Schlaeger says. Immunofluorescence staining and imaging techniques were used to observe such things as changes in cell number and marker expression over several days or weeks.
“We found that reprogramming is a dynamic process in which marker up- and downregulation occurs in a concerted manner over time, while the cells proliferate,” Dr. Schlaeger reports. “In rare cases, colonies of exponentially growing, fully reprogrammed cells appeared that were strikingly homogeneous. At the same time, we observed a large number of cells and small clusters or colonies of cells that remained frozen in partially reprogrammed states. Occasionally, we were able to directly observe conversion of such partially reprogrammed colonies to a more fully reprogrammed state. Our study allows us to identify in situ the rare live cells that are destined to undergo complete reprogramming.”
By using a variety of biomarkers and validating the study, Dr. Schlaeger and his lab were able to “distinguish the rare, fully reprogrammed colonies from incompletely reprogrammed colonies of cells with more limited developmental potential.”
PerkinElmer has a variety of instruments to image live cells, from the lab scale through high-throughput screening applications. “We use point scan confocal imaging in our imaging plate readers Opera and the just-released Operetta, which is best suited for long-term live-cell imaging with minimized cell damage,” according to Gabriele Gradl, Ph.D., global products leader, high-content screening, Bio-discovery business unit.
This technology can image cells for several days in low light conditions, helping researchers document such details as cell shape, proliferation, how cells differentiate, and the fate of cells over time, she explains.
“When working with live cells, maintaining their optimal environment is important,” Dr. Gradl says. Therefore, the cell::explorer has been designed to shuttle cells between the incubator and various instruments. “We’re focused on keeping cells in their native state,” emphasizes Achim von Leoprechting, Ph.D., vp and general manager, cellular imaging and analysis solutions, Bio-discovery business unit.
Fluorescence lifetime imaging microscopy (FLIM) is an additional, “elegant and robust technology to screen protein-protein interactions in live cells,” Dr. Gradl says. Opera confocal microplate imaging readers are available in a FLIM configuration. Designed for 24/7, high-throughput use, “it can sample up to 100,000 data points per day,” she adds. The smaller Operetta is designed for low- to medium-throughput labs.
For four-dimensional analysis, PerkinElmer recently introduced the UltraView VOX, which combines the high-speed image acquisition of the Volocity software with dual spinning disk hardware to double optical resolution, reduce laser power, and better protect the sample. “This also is being used to image small organisms such as C. elegans and zebrafish,” Dr. Gradl adds.
Cyntellect plans to launch its benchtop in situ cell-imaging platform (iSCIP) early this autumn for life science, drug discovery, and bioproduction applications. As yet unnamed, this system images the entire well, not just the center, using bright-field and fluorescence imaging. “This is important because samples don’t always evenly distribute, leading to inaccurate counts,” according to David Spector, director of business development. “This measures the entire well, right to the edge. This is a distinct feature. Others,” he notes, “have poor edge discrimination.” Bright-field imaging provides label-free analogs, eliminating dyes, washing, and some sample preparation. The fluorescence capability provides three fluorescent channels, offering a combination of excitation and emissions wavelengths. “Bright-field and fluorescence imaging may be combined for maximum culture assessment,” adds Gary Bright, Ph.D., senior director of applications development.
iSCIP can carry out direct counts as well as confluence measurements to determine general cell numbers. “To pick a common assay, cell health for example, iSCIP allows researchers to measure cell stress, toxicology, and apoptosis, measuring live, dead, and apoptotic cells, and how many are of each category,” according to Dr. Bright.