By Mike May
Recently, scientists watched a virus enter a cell–in real time. A protein called clathrin covered a spot on the cell membrane, which started to indent, making a vesicle that contained the virus, pulling it inside. Tom Kirchhausen–professor in cell biology at Harvard Medical School (Cambridge, MA) and in the program in molecular and cellular medicine and the immune disease institute at Children’s Hospital Boston, and one of the scientists who watched those viral particles–says, “This kind of imaging was a dream 10 years ago.”
FIGURE 1. A virus (red in upper images) is able to enter a cell through a clathrin-coated pit that facilitates internalization of the virus. (Imagery courtesy of Tom Kirchhausen, Harvard Medical School)
To fulfill such dreams, Kirchhausen and his team use an Axiovert 200M–an inverted microscope from Carl Zeiss MicroImaging (Thornwood, NY)–fitted with a spinning-disk confocal head from PerkinElmer Life and Analytical Sciences (Waltham, MA). “We can watch, in real time, events at the surface of a cell, and even look inside cells and tissues,” says Kirchhausen, “but doing that required stronger lasers and a better camera.” His system uses 50 mW, nanometer lasers from Coherent (Santa Clara, CA), and these get connected to the spinning disk with an acoustic optical tunable filter. Kirchhausen and his colleagues also use a computer-controlled, spherical aberration device–from Intelligent Imaging Innovations (3i, Denver, CO)–that corrects some distortions caused by the liquid environment. This device goes between the objective lens and a back-illuminated CCD camera, which provides improved quantum efficacy. With that overall system, says Kirchhausen, “We can get images in 20 ms.”
Microscope makers also seek improved speed in live-cell imaging. As an example, Nikon’s A1 confocal system can use its resonant scanner to grab images at 420 frames per second. “This works great for dynamic experiments, such as adding a drug to stimulate cells,” says Joel Silfes, senior biosystems applications manager at Nikon Instruments (Melville, NY). “But adding anything to a well makes a sample go out of focus.” The A1’s perfect focus system, however, refocuses on the sample 200 times a second. “So you don’t lose any data points,” Silfes says.
Broader access to imaging power
New technology also makes it possible for more laboratories to use live-cell imaging. For example, Nikon’s BioStation IM consists of a cell incubator and imaging system–including phase-contrast and fluorescent imaging, plus monitoring with a CCD camera. “We directed the Biostation IM at people who would love to take long-term, time-lapse images, but don’t have the knowledge and might not have the budget,” says Stan Schwartz, VP of Nikon Instruments. “This system takes care of temperature, humidity, carbon dioxide. With no microscope experience, you can get two-channel fluorescence and phase contrast superimposed in a movie that can run over two to three days.”
Beyond simplifying live-cell imaging, companies keep increasing how it can be used. As an example, researchers often want to manipulate live cells and then watch for changes over time. That’s just what Nanopoint’s CT-2000 allows. “Microfluidics control the environment–feeding the cells and keeping them alive without any physical change or manipulation–for up to five days,” says Brian Weatherly, director of sales for Nanopoint (Honolulu, HI). Here, the fluidics created the challenge. “For example,” says Weatherly, “the pressure inside a chamber must be precise, because it can disrupt the flow in and out of the well, and too much shear force will blow away a monolayer of cells.”
FIGURE 2. Merck scientists incorporate live-cell imaging with high-throughput screening in the company’s North Wales, PA facility. (Image courtesy of Merck & Co.)
Researchers also want to look at more kinds of live cells. “Stem-cell research, especially for human embryonic stem cells, has been flourishing everywhere except the United States,” says Ted Morris, who is the product marketing manager for research microscopy at Leica Microsystems Canada (Richmond Hill, ON). “Now, the U.S. market is also growing, since the Obama administration lifted restrictions.” Although stem-cell research can be extremely advanced, many steps can be relatively routine–say, just looking at cells to see if they survive in particular conditions. Morris says that this sort of work draws many stem-cell researchers to the Leica DM IL LED, which he calls a “nice inverted microscope for routine lab work, like looking at cell cultures.” He adds, “This microscope can go from bright field to phase contrast or integrated-modulation contrast, and the microscope automatically adjusts the light level when changing between these technologies.”
Going live in drug research
Living cells also enhance pharmaceutical research. “Live-cell imaging gives a much more-detailed understanding of the mechanism of action of genes and small molecules in signaling pathways related to diseases,” says Jeremy Caldwell, executive director of automated biotechnology at Merck Research Laboratories (North Wales, PA). “In Alzheimer’s disease, for example, you can watch APP [amyloid precursor protein] get cleaved in a live-cell assay, and then you can screen for small molecules or genes that can be introduced into the cells that reverse or enhance that event.”
To put such images to work in discovering or developing new drugs, Caldwell and his colleagues face an analysis challenge. “We can screen millions of compounds in a week in a highly sensitive live-cell-imaging assay,” he says, “but it could take six months to identify the hits.” Finding those hits–the compounds that create the desired effect–requires looking through lots of pieces of a puzzle. For instance, researchers might look at the kinetics of cellular events, such as a cell internalizing a disease receptor or a transcription factor–a piece of the machinery that starts turning DNA into proteins–moving from a cell’s cytoplasm to the nucleus. The more that must be watched in a live-cell assay, the more time it takes to analyze the data. Nonetheless, pharmaceutical scientists know that they must look at more features to understand the basic biology of disease, as well as the interactions with potential drugs. “We are beginning to appreciate that you need to measure multiple events simultaneously to get high-quality data,” says Caldwell, “because the interplay of these events relates to the disease biology.”
So today’s live-cell technology extends from basic research to industry. As always, scientists want to observe nature as purely as possible–not perturbed, not delayed, just real-time images of life.