Editorial Article
Monday February 15, 2010
by Caitlin Smith
The power to see inside cells, inside organelles, even to see individual molecules, is growing at an alarming rate. Cellular imaging is changing rapidly, according to Scott Olenych, consultant at Carl Zeiss MicroImaging. “New techniques are becoming available to address the questions of researchers in new and different ways. These new techniques offer researchers a novel and sometimes unparalleled view of the inner workings cells. Keeping abreast of these new techniques and incorporating them into their ongoing research efforts is a major challenge for scientists.” Here are some new developments in cellular imaging to help you see your targets.
It’s all about the cells
One of the most useful tools in cellular imaging is the assay kit—everything you need to do a defined experiment type on a particular topic. Because they usually include pre-optimized reagents and protocol, assay kits for cellular imaging can save you time and troubleshooting, letting you get right to the science. Enzo Life Sciences offers an array of fluorescence-based live cell assay kits, covering topics such as cell death pathways; oxidative stress, antioxidants, and scavenger assays; cell cycle and transcriptional arrest assays; and toxicology assays. In fact, toxicology assays may have a new impact on our health. “The importance of mitochondrial toxicity testing in drug development has recently come to the forefront,” says Wayne Patton, CSO at Enzo Life Sciences. “A diverse collection of approved drugs have subsequently been determined to be detrimental to mitochondrial function in humans, requiring them to either receive Black Box warnings from the FDA or to be withdrawn from the market altogether. Previous failures to predict hepatotoxic and cardiotoxic drug side effects has spawned renewed interest in monitoring mitochondrial status in living cells.” Enzo Life Sciences’ new Mito-IDT Membrane Potential Cytotoxicity Kit allows dynamic kinetic monitoring of mitochondrial energetic status by fluorescence microscopy or microplate-based cytometry. “The probe used in this particular assay is capable of detecting very subtle changes in mitochondrial membrane potential, being roughly 10-fold more sensitive than probes previously employed for this purpose,” says Patton.
It is crucial to keep your cells healthy before imaging (and during, for live cell imaging) to eliminate artifacts related to cellular stress or damage. Some cell types also are more sensitive to environmental conditions, such as primary cells. Tools such as the VivaView™ Incubator Microscope from Olympus can support happy cells during imaging, even over longer time periods. “Allowing for up to five different fluorescence channels, and acquiring with a cooled CCD monochrome camera, the system can easily be optimized for the most delicate, yet specific, research question,” says Stuart Shand, Olympus associate product manager. “In addition, the liquid handling option allows drugs or reagents to be added to the cells without disrupting the environment, a feature that is essential for minimizing temperature and atmospheric shock to the cells’ environment.” The latest VivaView includes hypoxic capabilities for experiments such as stem cell protocols. “Newly designed 10x, 20x, and 40x objectives, with phase contrast, provide a larger field of view and greater working distance,” adds Shand. “The 20x and 40x objectives options, with differential interference contrast (DIC), provide flexibility in high-resolution imaging.”
Imaging large and small
Sometimes one needs to image large fields of view one moment, then details of single cells the next. Systems that can provide multiple magnifications, as well as automation and high-content analysis, are increasingly in demand. GE Healthcare’s IN Cell Analyzer 2000 offers such imaging flexibility as well as many new features, such as rapid scanning to preview a selected area before acquisition; whole-well imaging to capture rare events; and a wide range of objectives (2x - 100x, plus high NA options) for different sensitivities. “Exciting developments are the ability to image whole tissues right through to subcellular organelles with one instrument, and to combine imaging with more predictive cell models, bringing considerable advantages in application areas such as toxicity,” says Tracey Zimmermann, communications leader at GE Healthcare.
Real-time and super-resolution
Imaging live cells has the advantage of observing cellular events in real time. “Zeiss’s VivaTome system is capable of delivering the fast frame rates required to capture dynamic biological processes,” combining the speed of a spinning disk with the efficiency of structured illumination, says Olenych. Photosensitive indicators offer a useful tool for molecular interactions. “Zeiss’s new DirectFRAP laser manipulation slider allows laser photomanipulation of chromophores in living cells. This allows researchers to perform Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP) experiments as well as photoactivation, photoswitching, and photoconversion of optical highlighter fluorescent proteins. The DirectFRAP slider can be added to widefield imaging systems, the Cell Observer spinning disk or the Laser TIRF3 systems,” says Olenych.
An emerging area in cellular imaging is super-resolution microscopy, which refers to imaging below the optical diffraction limit of a microscope (i.e. 200 nanometers). “The new Zeiss ELYRA system is designed to make super-resolution microscopy accessible for all researchers,” says Olenych. “The name ELYRA is a reference to the star Epsilon Lyrae which appears to be a single star, but when viewed with binoculars is revealed to be two stars. When viewed with even greater magnification the two stars are revealed to be two pairs of stars. Epsilon Lyrae is actually four stars—where initially only one was visible. The example of Epsilon Lyrae is an example of the type of advancement super-resolution microscopy represents to researchers using cellular imaging: the ability to see what was previously invisible.” Zeiss’s super-resolution systems include the ELYRA P.1 (designed for photoactivation light microscopy), the ELYRA S.1 (designed for super-resolution structured illumination microscopy, or SR-SIM), and the ELYRA PS.1, which combines both techniques.
Nikon’s new super-resolution microscope system, N-STORM, combines stochastic optical reconstruction microscopy (licensed from Harvard University) with Nikon’s Eclipse Ti research inverted microscope. “N-STORM provides dramatically enhanced resolution that is 10 times or better than that of conventional optical microscopes and will be capable of multi-spectral two-dimensional and three-dimensional nanoscopy, with lateral resolution to approximately 20nm and axial resolution to approximately 50nm, extending the role of the optical microscope to near molecular level resolution,” says Stan Schwartz, vice president at Nikon Instruments. “‘STORM’ is a new technology that reconstructs high-resolution fluorescence images (2D or 3D) from localization information of fluorophores detected with high accuracy and calculated from multiple exposures. It generates much more information and goes one step further, from structural to molecular understanding of the specimen.”
Another new Nikon system, the N-SIM, can yield nearly twice the resolution of conventional microscopes by combining structured illumination technology (SIM) technology (licensed from University of California, San Francisco) with Nikon’s Eclipse Ti inverted microscope and CFI Apo TIRF 100x oil objective lens (N.A. 1.49). “N-SIM also delivers the fastest imaging capability in the industry, with a time resolution of 0.6 sec/frame,” says Schwartz. “The newly developed TIRF-SIM illumination technique enables total internal reflection fluorescence (TIRF) observation with higher resolution than conventional TIRF microscopes and gives more detailed structural information near cell membrane. The new 3D-SIM illumination technique has the capability of optical sectioning of specimens, enabling the visualization of more detailed cell structures at higher spatial resolutions up to 20um in thickness.”
The new decade will bring new imaging techniques—and even more new data to organize and analyze. “In the future,” says Olenych, “I expect to see new developments that expand upon super-resolution microscopy, allowing sub-diffraction limit imaging to even greater resolutions and new techniques allowing high resolution imaging in living organisms.”