Bringing Living Cells Into Focus: A View of Inverted Microscopes

Date: March 30, 1998 Author: Jim Kling Tables of Vendors What's really going on here? That question used to puzzle bleary-eyed microscopists as they stared at slides of immobilized cells--dead cells, of course. Then along came the inverted microscope. Its unique design placed the light source above the sample and the magnifying objective below it, allowing these new microscopes to peer into live cells bathed in media. Suddenly, scientists had a new view of the neighborhoods and boroughs occupied by microtubules, vacuoles, and all the other cellular structures. Zeiss: Immunofluorescence of human skin cells, triple fluorescence; triple exposure with the single bandpass filter sets cytokeratin filaments (FITC), desmosomes (Texas red) and DNA (DAPI). Increasingly, cell biologists are studying live processes, "the inverted microscope lends itself to observing live material because [cells need] to be in medium or they need to be perfused," says Reinhard Enders, senior technical marketing manager at Olympus America, Inc. (Melville, N.Y.). In the late 1980s, researchers began taking a closer look at how cells respond to changes in their environment (in the form of added drugs, toxins, hormones, and other molecules). Inverted microscopes allowed researchers to observe the many aspects of cellular processes and responses. Electrochemical signals drive many cellular processes, and can also be a good indicator of others. To study these particular phenomena, electrophysiologists spend much of their time securing miniature electrodes (called patch clamps) to living cells. Once the realm of electrophysiologists alone, patch clamping now has found its way into biotechnology and the drug industry, as researchers examine cell permeability, drug sensitivity, and cellular transport using the technique. Because inverted microscopes afford more space above the sample than traditional upright microscopes, they have become a favorite tool in the field. "New technologies in molecular biology, micromanipulators, microinjectors, transgenic organisms, cel-lular recording, patch clamping, . . .[are] going to be the future explosion of biomedical science," predicts Andy Lee, manager of business development for Leica Inc.'s (Deerfield, Ill.) re-search microscopy division. "These trends in live cell research will continue to demand more and more of microscopes, both upright and inverted," he adds. Leica: Microinjection with LEICA DM IRB and Micromanipulator M. Photo courtesy of Dr. Jouni Uitto, Thomas Jefferson University, Philadelphia, Pa. But use of the instrument isn't limited to basic research. The red-hot field of in vitro fertilization is a "hugely important" application of inverted microscopes, says Stan Schwartz, manager of the biosciences department at Melville, N.Y.-based Nikon, Inc. The in vitro fertilization technique offers infertile couples new hope by manually docking a sperm cell with an egg and then reimplanting the now-fertilized egg cell into the woman to be carried to term. The tricky part is manipulating the sperm and egg so that they can merge. The inverted microscope answers that challenge by providing more space above the sample than does a traditional microscope, allowing room for micromanipulators and other instruments. As research delves deeper into life processes and live cell research, "the market for the upright microscope seems to be growing at a slower pace compared to the sales of inverted microscopes," Lee maintains. As one would expect, companies continue to develop new products and accessories to meet changing research demands. "We find that researchers are really very inventive in adapting microscopes to their needs," says Enders. In fact, all four of the major vendors stressed the continuing need for incorporating flexibility into instrument design. Take imaging ports, for example. The days of taking 35 mm snapshots and going home happy are passing, says Lee. "People want different levels of sophistication." A press of a button might capture that 35 mm slide, but researchers may want to be able to do photon counting or confocal microscopy at the same time. Olympus: COS cells infected with adenovirus containing modified green fluorescent protein. Image recorded without fixation 24 hrs. after infection. Dual port accessories allow scientists to perform more than one task at a time, such as splitting the image by spectral region. This is useful because it permits "ratio imaging," the determination of intracellular ions or pH with single excitation/dual emission fluorescent indicators. Fluorescence ratio imaging (FURA-2), producing green fluorescence emission, can also be combined with transmitted light in an other spectral region (e.g. red). In this way, the process being studied can be seen in relation to its cellular surrounding. Several other applications require more than one imaging port for simultaneous use of several specialized cameras or detectors. For example, additional ports empower the use of a highly sensitive camera (cooled CCD or SIT camera) for low light level fluorescence capture along with a less sensitive but high resolution digital camera. Fortunately, with a couple of models supporting up to four ports, even the most ambitious of users can find a suitable instrument. With repetitive strain injury (RSI) gaining increased attention as a health hazard, not just for writers (wince) but for anyone repeating the same task over and over, microscope companies are paying increased attention to the ergonomic aspects of design. "One major [consideration] in microscope development is how comfortable it is. How do you make a microscope that will do well in all of those operational environments? To do that is not trivial," says Becky Hohman, product marketing manager for light microscopy at Carl Zeiss, Inc. (Thornwood, N.Y.). So, be sure to test drive those models before you invest in them. Today's inverted microscopes come in three flavors. Each of the major companies offers less costly models tailored to less demanding uses. Typical features include bright field and phase contrast capacities, but not many bells and whistles. Research-class microscopes occupy the other end of the scale, with price tags roughly four times those of clinical instruments. Phase contrast is the typical imaging method, along with differential interference contrast. All offer fluorescence imaging as well. That's a crucial feature, says Enders, because fluorescence is such an effective tool. The green fluorescent protein (GFP), for example, can be linked to promoters in a cell line or animal model, producing fluorescence when that promoter is activated. "GFP allows you to study gene expression without interrupting the natural cell or growth cycle," says Enders. A third class, newly evolving, claims a middle ground between clinical and research microscopes. These instruments offer a little more "umph", as Lee puts it, than the simple (basic) microscopes, without breaking your budget. These microscopes typically offer all the features of clinical models but have added capabilities such as fluorescence. Of the four companies surveyed, only Nikon does not offer a model in this class, but the company plans to introduce one once "we get a chance to look at what the customer appreciates about those models... [then] we will build the most efficient, most feature-packed inverted microscope we can build," says Schwartz. Nikon: Drosophilia embryo captured on film using differential interference contrast (DIC) at 20x. Photo courtesy of Mel Brenner. With increasing numbers of features (typically) comes a more stable base. The stability of the microscope is a particularly important consideration in applications such as in vitro fertilization and patch clamping, where movement of micromanipulators and other tools can wreak havoc on attempts to manipulate cellular structures. If you get a chance, don't hesitate to load up the microscope with whatever equipment you'll be using, then handle it for a while to see how the microscope responds. Stability isn't just good for your experiment; it's good for your nerves. Molecular biology and microbiology continue to bring the world of living cells into clearer focus, and inverted microscopes figure to be the principal tools used to observe cells into the foreseeable future. New models with more accessories and flexible designs will continue to provide options to meet just about every research need. Shop carefully. Don't lose focus. The live cell research you save may be your own. Jim Kling is a writer and consultant based in Bellingham, Wash. He can be reached at jkling@nasw.com. Tables of Vendors

Bringing Living Cells Into Focus: A View of Inverted Microscopes

Interested in reading more?

Become a Member of