New forms of microscopy, supported by traditional assaying techniques, give life scientists ever more detailed views of the dynamic interiors of cells.

by Peter Gwynne and Gary Heebner

The old saw that seeing is believing has particular resonance for cell biologists. Ever since 1677, when Dutch biologist Anton van Leeuwenhoek used a simple light microscope to discover protozoa, blood cells, and other single cell organisms, researchers have relied on the evidence of their eyes to confirm their theories and suggest fresh approaches. In recent years, increasingly powerful means of visualization have provided more detailed views of cellular interiors. Forms of microscopy that reveal events at the molecular level, coupled with stains and probes and cell biology kits and reagents, have enabled researchers to make key discoveries about the inner workings of cells.

The technological improvements have emerged at an appropriate time. “Cell biology is in a revolutionary phase during which great leaps of understanding can be achieved,” says Barbara Armbruster, assistant product manager, biological transmission electron microscopy at JEOL. “The ultrastructural information we have about isolated proteins and macromolecular assemblies has to be placed in the context of intact cells and organs.”

Doing so requires two broad classes of technology. “The integrative effort,” Armbruster continues, “absolutely requires structural tools like electron microscopy to visualize the organization of cells directly at resolutions adequate to delineate macromolecules in situ.” However, traditional techniques of biochemistry continue to play a critical role in research. “What you can see with microscopy is, however, always dependent on the quality of the biochemical or immunological reagents you use,” explains Frank Neumann, head of new business development and technical services at Alexis Corporation. “Many of the actions inside the cell are performed by proteins; antibodies provide a way to detect these proteins,” adds Keith Watling, director of cell signaling research products at Sigma-Aldrich Corporation. “Whether the proteins are involved in cell structure, growth and development, or apoptosis, all are identifiable via antibodies.”

New, Improved Microscopy
More than 300 years after van Leeuwenhoek’s first look at cells, the light microscope remains a key tool for cell biologists. Manufacturers such as Carl Zeiss, Leica Microsystems, Nikon, and Olympus design and produce light microscopes capable of resolutions of 0.2 micrometers, as well as fluorescence microscopes, for research and clinical use. In recent years, a series of new, improved microscopic methods that stem from advances in physical science has reached the cell biology lab.

Three-dimensional confocal laser scanning microscopy (3-D CLSM) uses lasers, fluorescence, and computer power to obtain high-resolution images and three-dimensional reconstructions of cells. “Today the laser confocal microscope is the most important instrument for high level fluorescence microscopy,” says Martin Hoppe, vice president, marketing and sales for confocal microscopy at Leica. “Everybody wants to have one.”

The technology involves expanding a laser beam to make optimal use of the optics in the objective. A confocal aperture in front of the photodetector blocks any out-of-focus fluorescent light emitted from the specimen. The device generates a two-dimensional image of the specimen by performing a raster sweep of the specimen at that focal plane. As the laser scans the specimen, the system detects an analog light signal and converts it to a digital signal. The relative intensity of the emitted fluorescent light corresponds to the intensity of the image. A 3-D construct of a specimen can be generated by accumulating consecutive two-dimensional optical images.

The method has particular application to studies of living cells that other new forms of microscopy can’t manage. And increasingly, Hoppe says, “The technology is moving from pure imaging into analytical biology.” Thus, researchers can use the confocal microscope’s laser beams to manipulate specimens – by bleaching parts of a specimen, for example, to gain insights into diffusion at the cellular level. “It has also become very important for proteomics,” Hoppe adds. “Besides making beautiful 3-D images, you can focus on protein interactions.”

Leica offers several confocal microscopes. The range includes a universal system for 3-D resolution and analytics, a stripped down “workhorse system” targeted at small scientific teams, and a high-speed system, introduced last year, dedicated to live cell imaging. For analytical biology, new methods such as diode laser fluorescence lifetime imaging and fluorescence correlation spectroscopy can be combined with the core instruments. The company emphasizes user-friendliness. “Forty percent of our R&D work goes into making the systems easier to use,” Hoppe says.

A Very Powerful Tool
At a finer level of visualization, cell biologists have recently come to rely on electron microscopes. “Five years ago, electron microscopy was a dying art in the life sciences,” says Cameron Ackerley, director of electron microscopy in the department of pediatric laboratory medicine at Toronto’s Hospital for Sick Children. “Then life scientists rediscovered that they could identify a lot of things on the basis of sheer ultrastructures. Used in conjunction with highly specific immunocytochemical techniques such as immunogold labeling, the electron microscope has become a very powerful tool in trying to understand the functionality of a whole slew of gene products.”

Based on the interaction of electrons with components of tissues or cells and electron dense stains, electron microscopes can provide resolutions of 0.1 nanometers. That permits cell biologists to determine structural details down to molecular dimensions in cells and cellular components. Two basic types of microscope exist. Transmission electron microscopy (TEM) involves the passage of electrons through a stained ultrathin section of material. Scanning electron microscopy (SEM), by contrast, provides a three-dimensional image of a specimen’s surface.

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In both cases, preparation of the specimen must ensure that its structure appears under the microscope just as it does in a living cell. That’s difficult in the case of TEM, which uses slices of biological objects or suspensions of protein molecules, viruses, and the like. Recently, however, vendors such as Hitachi and JEOL have developed a cryonic approach to this type of electron microscopy, allowing studies to be conducted with specimens maintained at liquid nitrogen or liquid helium temperatures. “Dynamic events are captured at the millisecond level with rapid freezing of specific functional states, providing high-resolution detail and contextual structural information in the same photomicrograph,” JEOL’s Armbruster explains. In addition, says Jaap Brink, JEOL’s biological applications manager, “Applying TEM to biological specimens labeled with atoms such as gold provides a way of monitoring protein interactions in the cell.”

High Resolution and Magnification
JEOL also offers research grade SEMs with resolutions of 1.0 nanometers and maximum magnifications of 650,000 times. To date, this technology has had limited appeal for cell biologists. “Field emission SEM was very useful for immunocytochemistry of surface antigens, but not much use for cell interiors as you were limited to cell membranes,” says Ackerley. “To identify internal ultrastructure, cells had to be permeabilized with strong detergents or fractured at liquid nitrogen temperatures and macerated to remove cytosolic components that would otherwise obscure surface detail.”

However, that’s changing. “User-friendly cryostages interfaced to cryopreparative chambers external to the column have been developed that allow investigators to freeze, fracture, and sublimate surface water exposing cellular architecture, render it conductive with a thin layer of metal, and transfer it to the cryostage without exposing the specimen to atmosphere,” Ackerley explains. That allows researchers to examine cells and tissues in a frozen hydrated state. Using this technology, cells and macromolecules can be examined without exposing them to any fixatives or solvents that might leach or displace important constituents. The methodology also opens the door for the use of quantitative analytical electron microscopy as both extracellular and intracellular components remain intact, unlike the ultrathin cryosection used in analytical TEMs. “There’s also hope for wet live cell imaging in the SEM using environmental chambers that retain atmosphere in the chamber but are electron lucent,” Ackerley continues. “Images can be obtained using either filtered backscatter or conventional backscatter electron imaging.”

A Closer Look
For an even closer look at cellular components, such as two-dimensional crystal membranes, down to subnanometer resolutions, researchers can turn to atomic force microscopy (AFM). An atomically sharp tip hanging from the end of a cantilever sweeps over the surface of a specimen. The scanning procedure generates a signal that is used in a feedback loop to maintain the tip at a constant force (to obtain a 3-D height image of the sample), or a constant height (to obtain force information) in relation to the sample’s surface.

Developed in the 1980s, AFMs had the primary purpose of measuring the roughness of surfaces at the atomic scale. But since the instruments can detect images on nonconducting surfaces, biologists started to apply them to such tasks as analyzing small molecules such as DNA and proteins and organic monolayers. “I’m certainly seeing a lot more biological applications, particularly as the technology can work with transparent fluids and at physiological temperatures,” says Charles Mooney, JEOL’s assistant product manager for scanning probe microscopes. “The advantage of AFM is that you can image your living cells in controlled conditions, allowing you to monitor dynamic events,” adds Sophia Hohlbach, biological applications scientist in the metrology division of Veeco Instruments. Veeco has patented a method of intermittent contact, called TappingMode, that permits scientists to run the AFM over a surface without applying a frictional force and minimizing the interaction force.

Cell biologists have applied the AFM’s unique capabilities to study the dynamic behavior of living and fixed cells such as endothelial cells, cardiac cells, red and white blood cells, bacteria, platelets, and other types of cells. JEOL, for example, has developed a freeze fracture method that enables AFM to look at red blood cells.

With a closed loop system, scientists can use an AFM as a nano-manipulator to position the cantilever onto specific locations in a cell membrane. “It gives you a way to get information about the local elastic properties as well as stimulate a cell locally,” says Jens Struckmeier, project manager for force microscopy in Veeco’s metrology division. “It will become increasingly more important in cell biology.”

The company has also developed a PicoForce SPM system to investigate individual proteins. “One useful application is looking at transmembrane proteins,” Struckmeier says. “That enables you to map both the structure and the energy landscape of individual proteins within the membrane.”

AFM has one added advantage: It works synergistically with other microscopic methods to facilitate research in cell biology. JEOL and Veeco, for example, offer a combination of AFM and scanning tunneling microscopy, another form of scanning probe microscopy. “You can also combine AFM with fluorescence and confocal microscopy,” Struckmeier says.

Adding Antibodies
Even in combination, though, microscopes can reveal only limited details about the inner workings of cells. Cell biologists still need to complement their microscopy measurements with traditional biochemical assays – particularly those that involve antibodies. “The biggest advantage of antibodies is specificity,” says Mike Ignatius, R&D director of the cell biology group at Molecular Probes, a subsidiary of Invitrogen Corporation. “High quality and specificity are absolutely paramount in antibodies,” agrees Watling of Sigma-Aldrich. “There are a lot of poor quality antibodies out there, brought to market sooner than necessary. We pride ourselves on putting out high-quality antibodies. You need them to be completely specific to what you’re trying to measure.”

Antibodies have particular value in the study of kinases, the proteins that control many cellular functions. The production of targeted antibodies that identify the active form of protein kinases has given researchers better understanding of kinases’ roles in signal transduction pathways. BD Biosciences Pharmingen, Chemicon, and Sigma-Aldrich, among other companies, offer antibodies for this general area of research, which is gathering momentum. “Our new catalog that will come out next year will have over a thousand new products for cell signaling,” says Sigma-Aldrich’s Watling. “We have made 600 new products this year, including antibodies and nonantibody based reagents,” adds Ignatius of Molecular Probes. Other companies that offer assaying products for cell biology and related subdisciplines include Alexis, EMD Bioscience (formerly Calbiochem), ICN Biomedicals, Sigma-Aldrich, and Tocris Cookson.

‘The Dyes That Bind’
Antibody based probes are ideal for identifying specific cell populations on the basis of differences in their cell surface proteins or markers and for identifying and locating subcellular structures. Companies such as Midland Certified Reagents and Sigma-Aldrich are primary manufacturers of the stains and dyes used in these detection systems. And Molecular Probes provides many of the fluorescent labels used with antibodies, including the entire line of stable and bright Alexa Fluor dyes and the new Zenon labeling reagent for antibodies.

“We also make many nonantibody based fluorescent molecules used in the interrogation of cells,” says Ignatius. “These ‘dyes that bind’ are selective for most subcellular targets and many are fluorogenic, glowing only when bound to their targets. For example, we have reagents selective for RNA or DNA inside live cells, organelle specific dyes, and ion sensitive dyes. The beauty of these is that they are all small molecules, are cell permeable, and work on live functioning cells, in most cases by the simple addition to the media bathing the cell with no subsequent washing. ‘No-wash’ detection reagents are ideal for the emerging field of high content screening.”

Alexis, meanwhile, has begun to offer a cell permeable far red fluorescent DNA dye called DRAQ5 for multicolor analysis. “It’s the ultimate dye for fluorescence microscopy,” says Neumann. “The unique thing about this photostable DNA-specific dye is that you don’t need an ultraviolet laser to excite it. You can perform analysis simultaneously in combination with additional conventional red or green labels with no need to compensate or change the exciting light source. You can excite it from a common 488 nm laser that most labs have. If you’re monitoring events in living cells, you don’t want them under an ultraviolet light just to stain the nuclei.”

Continued advances in cell biology facilitated by new forms of microscopy and assaying hold great potential for basic scientific understanding and the treatment of disease. The tools and techniques under development by researchers and vendors promise discoveries that will add to knowledge of the ways in which cells function.

WEBLINKS ADVERTISERS American Type Culture Collection (ATCC) diverse materials (such as microorganisms, cell lines, and recombinant DNA materials) to media and other reagents, as well as bioscience laboratory services 703-365-2700 www.atcc.org BD Biosciences Pharmingen products for immunology, apoptosis, cell biology, neurobiology, and molecular biology research 858-812-8800 www.pharmingen.com CELLnTEC Advanced Cell Systems epithelial cells, specialized cell culture media, and services for life science researchers +41 (0) 31 631 25 08 www.cellntec.com Genetix [UK] laboratory automation products and services for microarrays and sequencing +44 1425 624 600 www.genetix.com Genetix [USA] 877-436-3849 Quantum Dot Corporation quantum dots used for the detection of nucleic acids and proteins 510-887-8775 www.qdots.com SANYO Sales & Marketing Corporation / SANYO Electric Biomedical Co., Ltd. constant temperature equipment (incubators, ovens, refrigerators, and freezers) www.sanyo-biomedical.co.jp Upstate Biotechnology products, technology platforms, and services based on cell signaling for life science research and drug discovery 781-890-8845 www.upstatebiotech.com FEATURED COMPANIES Alexis Corporation signal transduction kits and reagents www.alexis-corp.com AAAS (American Association for the Advancement of Science) nonprofit scientific society www.aaas.org/meetings BD Biosciences Pharmingen antibodies for signal transduction research www.bdbiosciences.com Carl Zeiss imaging detection systems, microscopes www.carlzeiss.com Chemicon International, Inc. antibodies for signal transduction research www.chemicon.com EMD Bioscience (formerly Calbiochem) signal transduction reagents www.emdbiosciences.com Hitachi High Technologies Corporation electron microscopes www.hitachi.com Hospital for Sick Children hospital www.sickkids.on.ca ICN Biomedicals signal transduction reagents www.icnbiomed.com JEOL-USA, Inc. electron microscopes www.jeol.com Leica Microsystems, Inc. imaging detection systems, microscopes www.leica-microsystems.com Midland Certified Reagent Company, Inc. stains and dyes www.mcrc.com Molecular Probes (a subsidiary of Invitrogen Corporation) fluorescent markers and probes www.probes.com Nikon Corporation imaging detection systems, microscopes www.nikon.com Olympus Corporation imaging detection systems, microscopes www.olympus.com Sigma-Aldrich Corporation antibodies, kits, and reagents www.sigma-aldrich.com Tocris Cookson, Ltd. signal transduction reagents www.tocris.com Veeco Instruments atomic force microscopes www.veeco.com

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