Fluorescence microscopy is to light microscopy as Las Vegas is to Poughkeepsie, piercing the darkness under the scope with brilliantly colored light given off by the samples themselves. Fluorescent microscopy offers a high degree of sensitivity, as well as the ability to accurately zero in on the cell or molecule of interest.
Hundreds of fluorescent dyes have been developed for use in microscopy, covering a generous range of wavelengths of excitation and emission. The technique has become a mainstay of microbiology, molecular biology, and cell biology, where images of brightly glowing cells have graced the pages of every scientific journal. More recently, new techniques for visualizing live cells have generated a great deal of excitement and interest in the field. As the technology for fluorescently labeling cells and samples has advanced, instrument vendors have responded by improving and upgrading the instruments, and not only the microscope, but the optics, cameras, filters, and analytical software as well.
Improved Fluorophores
Protein fluorophores offer the option of “built-in” fluorescence. Their use has exploded in popularity, especially in fluorescence resonance energy transfer (FRET) experiments, where one fluorophore transfers a photon to another in close proximity. Molecular Probes (Invitrogen) has recently introduced a FRET-based sensor called Cameleon, which links a cyan fluorescent protein to a yellow fluorescent protein through calmodulin. When the calmodulin encounters calcium ions from a GPCR or ion channel, it undergoes a conformational change, transferring some of its emission light from cyan to yellow. This concept is not new; however, new technology for delivery and expression makes it easier to plan and execute experiments using these principles.
Molecular Probes has packaged the gene for Cameleon and other proteins into baculovirus vectors, creating ready-to-use genetically encoded reagents for cellular studies. Baculovirus has recently garnered interest in academic and drug discovery labs as an inert delivery vehicle to mammalian cells (since mammalian cells will admit the baculovirus and allow it to deliver its genetic payload without developing true infectivity). Although the Cameleon system has many advantages, it may not be right for every calcium-sensing application. Says Magnus Persmark, senior product manager at Molecular Probes/Invitrogen: “We are not suggesting that researchers should jettison all the tools they have gotten used to. We are enabling them to do more than they hitherto could have done by giving them a larger toolbox.” Infrared fluorescent dyes are also becoming increasingly important because of the interest in live cell imaging. Wavelengths in the infrared range are less damaging to live cells than, for example, ultraviolet light.
Once you've chosen a fluorophore, and a method for staining your sample, the next step is choosing a microscope platform. The choice is no longer as straightforward as it once was.
The Right Microscope for the Job
In the past, fluorescence microscopes have been used to acquire good quality two-dimensional images of the sample. Now, it's possible to capture images in real time (as opposed to the delay generated by standard confocal microscopes), view sections of the sample, create a three-dimensional image, or look at a surface such as a membrane. Choices in instrumentation are now very much customized to the type of application. Says Chris Vega, PhD, product marketing manager for Leica Microsystems: “Depending on the customer's application, whether they're working with live cells or stained specimens on a slide, there are different instruments to suit those purposes.”
The Leica TCS SP5 confocal microscope is ideal for imaging rapid cellular processes. The filterless SP spectral detection system offers high efficiency with up to five simultaneous output channels. Leica's total internal reflection (TIRF) microscope offers improved live cell imaging with very high axial resolution. TIRF is a particularly useful imaging technique for visualizing vesicle transport, interaction between molecules, and membrane dynamics. The evanescent illumination field from the TIRF objective penetrates specimens only a few nanometers, resulting in very high signal-to-noise ratios at the plane of focus. One of its primary applications is the study of transport through cellular membranes. On the horizon for Leica is a system called STED, which breaks barriers for X and Y axis resolution in microscopy. Says Vega: “This is really going beyond what is classically determined to be the limit. We can see more resolution than what people thought was possible.”
Olympus's two-photon excitation microscope is its flagship live cell imaging product. Two-photon excitation is based on quantum optics concepts, and gives the advantages of eliminating background signal and increasing the depth of penetration two- to three-fold over a standard confocal microscope. “The two photon technique allows us to image 2 to 5 fold deeper than a standard confocal, and we have two photon images that were collected over 700 microns deep,” says Nicolas George, product manager for research microscopes at Olympus. The deep penetration is actually a combination of the properties of two-photon excitation and the use of low energy infrared light, which minimizes light scattering.
Making Connections
Simon C. Watkins, PhD, is a professor at the University of Pittsburgh and director of the popular Quantitative Fluorescence Microscopy workshop out of Mount Desert Island Biological Laboratory in Salsbury Cove, Maine. The workshop, which emphasizes practical methods in fluorescence microscopy, has generated so much interest that they turn away two prospective students for every filled slot. “The thing that most people want to do now is live cell microscopy. You take what is traditionally dead material and look at it live,” explains Watkins. The attraction of live cell microscopy over traditional mounted and stained slides is analogous to the advantages of live moving pictures over photographs. Movies allow you to see a sequence of events, rather than a frozen moment in time.
It was the use of live cell fluorescence microscopy that led Watkins and his collaborator, Russell D. Salter, to the discovery that dendritic cells communicate calcium fluxes by means of tunneling nanotubules—a direct, physical network of tubes running between distant cells.1 These results raise interesting questions about the role of dendritic cells in the body.
Research in fluorescence microscopy is limited only by the limits of the technology, so the search for better instruments and reagents continues to have direct payoffs in the form of scientific discovery.
References:
1Watkins SC and Salter RD, “Functional connectivity between immune cells mediated by tunneling nanotubules,” Immunity, 23(3):309-18, Sep 2005 .
