Light Microscopy Enables Scientists to Peer Inside Cells In Real Time

Although the laws of physics dictate how much an object can be magnified and still clearly seen, scientists continue to expand their view of the microscopic world beyond the cellular level. New light microscopy methods and technology have made it possible for scientists to view previously undetectable tiny structures inside of cells, and to examine such objects in real time as cells carry out their activities. "New microscopy techniques, particularly those involving fluorescence microscopy and the new fluorescent probes that are being developed, have made it possible for researchers to do many different things that would have been totally impossible to do two years ago," says Fred G. Lightfoot, director of the Center for Microscopy and Image Analysis at the George Washington University Medical Center in Washington, D.C. Besides magnifying cells and other microscopic objects, scientists are now viewing subcellular organelles and making quantitative measurements of dynamic intracellular activities. In the 1960s, the electron microscope, in which beams of electrons form a magnified image, allowed researchers to see cells as never before-magnified millions of times with exceptional detail. While the electron microscope is still useful for many applications in biology and materials science, for life scientists, electron microscopy may go the way of the dinosaur. The harsh treatment necessary to prepare biological samples for bombardment with electrons kills cells, making the instrument unsuitable for imaging living cells. New ways of imaging have brought cell biologists back to their old workhorses-the light microscope. "We are today at a point similar to where we were in electron microscopy in the late 1960s," explains Lightfoot. Just as electron microscopists create thin slices, or sections, of tissues and cells before viewing, "this new wave of light microscope techniques allows us to create optical sections of living cells, and also allows us to view the dynamic changes that occur inside." "In the past 10 to 15 years there has literally been a revolution in the field of microscopy because of the development of techniques such as epi-fluorescence," agrees Jeff Travis, a cell biologist at the State University of New York, Albany. "Microscopes now provide images with better contrast and of much higher quality than ever before." ALL-PURPOSE SCOPE: Leica's DM LB can be transformed from a simple transmitted light microscope into a fluorescence system with full-image documentation. Industry pioneers-such as Carl Zeiss Inc. Microscope Division in Thornwood, N.Y.; Nikon Inc. Instrument Group, based in Melville, N.Y.; Olympus America Inc. Precision Instrument Division, also of Melville; and Leica Inc. of Deerfield, Ill.-are ushering in the wave of interest in light microscope applications with new optics and microscopy systems. These devices enable researchers to observe in new ways the cells they've been studying for years. While the ability to magnify the microscopic world is virtually limitless, the ability to distinguish fine details in enlarged images is not. Light microscopes, invented and first used to view bacteria and other types of cells in the 17th century by Antoni von Leeuwenhoek, reached their theoretical limit of resolution (the point at which two microscopic objects can be seen discretely) prior to the turn of the 20th century. The development of phase-contrast optics and differential interference contrast (DIC) microscopy in the middle of this century gave researchers their first opportunity to attain high-contrast images of living cells. Prior to phase-contrast microscopy, scientists studied dead cells fixed to slides and stained with colorful dyes to provide contrast. The development of epi-fluorescent illumination in the mid-1970s, as well as confocal optics, invented in 1955 and refined during the last decade, is widely credited with touching off the light microscopy revolution. "Without a doubt, the confocal microscope is the impetus behind the resurgence of interest in light microscopy that's occurred over the last four or five years," says Lightfoot. With a confocal microscope, scientists are able to observe cells in exquisite detail and image dynamic cellular events as they occur. "The confocal microscope gives researchers the ability to optically section cells and observe cellular activities in three dimensions," he adds. With all types of light microscopes, images are formed when light waves that travel through a specimen are collected by lenses. As the light waves pass through various regions of a cell, some are diffracted (bent) to varying degrees. When these waves are recombined to form the magnified virtual image, regions of light and dark (contrast) are created. In bright-field microscopy, living cells-essentially an aqueous solution bounded by a membrane-disappear because of a lack of contrast between the cell and its background medium. In phase-contrast and DIC microscopy, contrast is generated by altering how the system of lenses collect and recombine light waves. This creates a more contrasted image, rather than a higher degree of resolution. However, a drawback with conventional microscopes that collect light from a relatively wide area is the issue of depth of field. "With a conventional wide-field microscope, the image formed when you focus is actually an integration of images from various depths in the specimen," explains Travis. The light coming from other parts of the specimen creates an out-of-focus "haze" that can obscure detail in the desired image. Epi-fluorescence illumination was developed to overcome the limitations imposed by the physical properties of light. A fluorescence microscope, for instance, shares the same basic optical system of the light microscope, with the exception that only certain wavelengths of light, those that are of lower energy (higher wavelength), are detected. In fluorescence microscopy, incident light rather than transmitted light is used for forming images. "Epi-fluorescence utilizes a single objective lens and a dichroic beam splitter to filter out wavelengths of light that render an image out-of-focus, resulting in better contrast and image quality," explains Travis. Epi-fluorescence illumination is available with many microscopes, including the Axiovert (inverted) and Axioplan research microscopes from Zeiss, and Nikon's Optiphot-2, a multifunctional research microscope featuring Nikon's Quadfluor epi-fluorescence illumination system that can be configured for simultaneous fluorescence and DIC imaging. Epi-fluorescence used simultaneously with transmitted light techniques, such as phase contrast and DIC, provides investigators with qualitative and quantitative information about cellular structures and physiology. VERSATILE: The iX70, a model from Olympus America Inc.'s iX Inverted Microscope System The Olympus iX Inverted research microscope from Olympus America Inc. Precision Instrument Division can also be easily configured for both transmitted and incident light applications. In addition, investigators using the iX can choose up to five output ports for video or CCD (charge-coupled device) imaging, photomicrography, microphotometry, and for confocal laser-scanning microscopy. The Leica DM LB is an all-purpose microscope suitable for many applications in the research laboratory. The DM LB can be quickly transformed from a simple transmitted light microscope into a proficient fluorescence system with full-image documentation capabilities. Standard features include Leica's Delta optics with a 25 mm field of view, a ceramic stage and a three-gear focus drive. Images can be captured for posterity by attaching a camera to the microscope, and most manufacturers can adapt their microscopes to carry a detection system that might be simple (a still or video camera) or complex (a CCD camera or a photomultiplier tube, or PMT). The PZMT stereomicroscope and the H602T inverted research microscope from World Precision Instruments in Sarasota, Fla., are trinocular to provide researchers with the ability to capture their images on film. For each level of magnification, the quality of a microscopic image is directly related to the light-gathering capabilities of the objective lens, the lens into which light from the specimen enters first. This is particularly true in fluorescence microscopy, in which the light energy emitted from the sample is generally weak and hard to detect. NEW FROM ZEISS: Carl Zeiss Inc.'s Axiophot 2 photomicroscope, left, features a notebook PC-controlled photo module. The company's Axioplan 2 is a universal microscope. Optics such as Zeiss' Fluar ICS objectives are designed to provide maximum light-gathering potential for bright images over a wide spectral range in many applications, such as measuring intracellular calcium levels with the fluorescent dye fura-2 and observing living cells, where low light levels must be detected. Zeiss' Plan-Neofluar objectives also offer versatility-they are well suited for use in epi-fluorescence, phase contrast, DIC, and other microscopy techniques that can all be performed with the same set of objective lenses. Nikon's CF N Plan Fluor and Plan Fluor Phase optics are considered "universal objectives" because several applications-including DIC, conventional bright-field microscopy, phase contrast, and fluorescence-can all be performed using the same objective lens. Nikon's newest Plan Fluor lens is a 40X oil-immersion lens with a high (1.3) numerical aperture (a mathematical expression of the light-gathering abilities of a lens). The Plan Fluor is "a big photon [light]-gathering lens," according to Mel Brenner, technical manager of Nikon's Biomedical Division. "The small increase in the numerical aperture of this lens results in a large increase in its light-gathering capabilities," says Brenner. This is particularly important in microscopic studies in which the signal might be of very low intensity, such as in the cytogenetic technique of fluorescence in situ hybridization (FISH). In FISH, fluorescent probes bind to and label target sequences directly in cells or tissues. "Because individual fluorescent signals created by FISH and other fluorescent techniques are weak and difficult to detect, fluorescence images are much brighter with the Plan Fluor objectives," Brenner adds. FISH techniques are used by geneticists and cell biologists to localize DNA and RNA sequences on specific chromosomes in metaphase spreads (in which cells arrested at a certain point in their cell cycle spill their contents on slides), in cells, and in tissues. In FISH, DNA or RNA probes specific for regions on chromosomes are labeled with fluorescence and then added to preparations of chromosomes or cells, where they bind (hybridize) to regions similar in nucleotide sequence. Chromosomes bound by the labeled probe are thus tagged with the fluorescent dye, which is observed by fluorescence microscopy. Depending on the probes used, entire chromosomes, chromosomal regions, or even single-copy target sequences can be identified with this technique. In genetics and biomedical research, FISH can be applied to karyotyping by researchers in search of chromosomal abnormalities associated with genetic diseases such as Down's syndrome, or to study the chromosomes of tumor-derived cells for signs of a cancer-causing mutation. Cell and developmental biologists use FISH for mapping cell receptors and cytoskeletal proteins, to identify particular messenger RNAs, or to localize specific genes in cells. With a series of differently colored fluorescent labels, several distinct chromosomal features can be detected simultaneously. In addition to a fluorescence microscope, multicolor FISH requires special filters, and a sensitive detection system, such as a video camera or digital CCD. The use of a computer and dedicated software is also required to capture and correlate images at several different wavelengths, record the location of the chromosome on the slide, and analyze the collected information according to the interests of the investigator. Confocal optics, like epi-fluorescence, minimize the interference of out-of-focus light with the image-forming rays. When specimens are thick or heavily labeled with fluorescence, a confocal microscope can create optical sections of a specimen and permit an investigator to observe the various depths in focus. "Confocal microscopy is a completely different method of imaging," says Travis of SUNY-Albany. As opposed to the two-dimensional images produced by a conventional light microscope, confocal microscopy generates a three-dimensional view created a point at a time. With a confocal microscope, a highly focused beam of light scans the specimen in parallel lines from side to side. Transmitted or reflected light-or, in the case of fluorescence, light that is emitted-travels back through an aperture the size of the point on a pin before reaching a photodetector, often a PMT. Because only the refocused light is collected, out-of-focus light coming from other focal planes is effectively rejected. Point by point, the specimen is scanned in three dimensions, and the resulting images are combined into a single, three-dimensional view of the microscopic object. Because the light of a laser is quite cohesive, many confocal systems use this intense form of energy as the illumination source. CONFOCAL SYSTEM: Noran Instruments' Odyssey XL can be added to research-grade microscopes from various manufacturers. In actuality, a confocal microscope could be any type of light microscope to which a confocal unit is attached. "Confocal microscopy is simply a different method of illumination and detection based on the optical system of a light microscope," says Willy Hausner, North American sales manager for Noran Instruments in Middleton, Wis., a company that specializes in X-ray microanalysis, digital imaging, and confocal microscopy. Laser confocal microscopy used with fluorescent dyes combines the sensitivity of fluorescence with the ability of confocal microscopes to filter out background "noise" and create detailed 3-D images of cells. Confocal instruments are available from the major microscope manufacturers as well as from companies such as Technical Instrument Co. in San Francisco, which distributes the Nikon product line in addition to its own K2-S BIO real-time confocal scanning microscope system and the Bright i laser confocal scanning microscope. Noran Instruments' Odyssey XL confocal system can be added to research-grade microscopes from Zeiss, Nikon, Olympus, or Leica to create confocal images. The system is interfaced with an Indy workstation from Silicon Graphics Inc., which permits rapid acquisition of images from an infinite number of scan points in the cell volume. "Even at scan rates of up to 480 images per second, images are recorded digitally to the computer's hard drive, which provides the computational power to perform complicated imaging tasks, such as making measurements of volume and surface area in three dimensions," Hausner reports. The MRC-1000 confocal laser-scanning system is a computer-controlled confocal microscope system from Bio-Rad Laboratories in Hercules, Calif., that features new software for designing and saving experimental methods for recall at a later time. The laser is delivered through a fiber-optic cable, which removes vibrations that would normally occur as a result of the laser cooling fan, thus providing a more stable system. The newly designed PMT detection system in the MRC-1000 reportedly increases the sensitivity by a factor of at least two. The image-processing capabilities of the LSM 410 Inverted and LSM 310 confocal microscope systems from Zeiss give researchers the ability to perform photometric analyses of cellular topography and profilometry as well as ratiometric assays for changes in intracellular calcium ion concentration or pH. The LSM 410 Inverted microscope employs a combination of up to four lasers for exciting different color fluorescent dyes with three fluorescence PMT detectors, allowing researchers to record and display three different fluorescence signals simultaneously. COMPUTER-CONTROLLED: Molecular Dynamics Inc.'s MultiProbe 2010 Inverted Confocal Laser Scanning Microscope System The MultiProbe 2010 Inverted Confocal Laser Scanning Microscope System from Molecular Dynamics Inc. of Sunnyvale, Calif., features a computer-controlled means of confocal sectioning and three-dimensional reconstruction of images. Used with Molecular Dynamics' ImageSpace Confocal software, the CLSM 2010 allows investigators to perform a wide variety of functions, from measuring and analyzing 3-D volumes to generating publication-quality output. Although the theoretical limit of resolution of the light microscope defined by such parameters as wavelength and numerical aperture is not to be breached, new methods of imaging are allowing researchers to observe the microscopic world more clearly. "Theoretically, we are limited to the resolution of the optical system that we are working with, yet we can see things in more detail now than ever before," Lightfoot notes. "It's an exciting time to be in the field." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Light Microscopy Enables Scientists to Peer Inside Cells In Real Time

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