With Fluorescence Microscopy, Researchers See Cells In A New Light

Cells In A New Light By combining the sensitivity of fluorescent dyes with optical systems that can detect colorful but low-intensity fluorescent light, researchers in many life sciences are able to peer inside cells and view fine detail as never before. With a fluorescent microscope, an investigator is now better able to study individual cells and image subcellular entities, such as organelles, proteins, microtubules, and chromosomes. Owing to advances in fluorescent microscopy techniques, researchers have the ability to study the intracellular dynamics of living cells, as such events occur in real time. SEPARATES SIGNALS" Nikon's QuadFluor epifluorescence illuminator "Within the last 15 years, fluorescence microscopy has developed into one of the most important techniques in cell biology," comments Eric Gruenstein, a professor of molecular genetics and biochemistry at the University of Cincinnati Medical School and president of Cincinnati-based Intracellular Imaging Inc. "Two relatively recent developments have pushed fluorescence microscopy to the forefront of the available technology. First was the development of new fluorescent probes [that] allow us to look at specific events occurring inside of living cells. And second, with the aid of computers we are now equipped to analyze quantitative changes that occur in these cells." Fluorescence microscopy is based on the same principles of optics as light microscopy. To create an image in a light microscope, light waves from an illumination source pass through and around a specimen. Those light waves are gathered and then recombined by the lens system of the microscope to form the image of the specimen. In the case of epi-fluorescence microscopy, a single lens serves as both the condenser (the lens system that focuses the light before it reaches the specimen) and the objective. The light passes through the lens and excites fluorescent dye molecules in the specimen. The excited dyes re-emit light at a higher wavelength (a different color). Some of that light is collected by the objective lens and then sent to a detection system to produce a fluorescent image. Achieving a fluorescence image is optically demanding, because the low-energy light emitted from a fluorescently stained object is generally not as intense as the high-energy wavelengths initially used to excite the dye. Accordingly, the optical system of a fluorescence microscope incorporates one or more filters that fine-tune the light going to and coming from a specimen. "If you used a blue light to excite a green dye and allowed the blue light to come through the ocular lenses or detection device, the blue light would overwhelm the lower-energy green light coming from the specimen," explains Paul Millman, vice president for sales at Chroma Technology Corp. of Brattleboro, Vt. Chroma manufactures filter sets for microscopes made by Nikon Inc. of Melville, N.Y., and other companies. Chroma filters are specific to the various dyes used for fluorescent imaging. Selected Vendors of Fluorescence Microscopy Equipment and Supplies Applied Precision Inc. Chroma Technology Corp. Intracellular Imaging Inc. Leica Inc. Meridian Instruments Inc. Molecular Dynamics Molecular Probes Inc. Nikon Inc. Olympus America Inc. Oncor Inc. Scanalytics/CSPI Carl Zeiss Inc. "To extinguish the background 'noise' from the true signal, filter sets optimize the wavelengths of both the light used to excite the dye and the emission signal," Millman says. The QuadFluor epi-fluorescence illuminator from Nikon accepts up to four fluorescence filter cubes for separating different fluorescent signals, or to image several fluorochromes simultaneously. Nikon's Optiphot-2 is a multifunctional research microscope that can be equipped with the Quadfluor illuminator. The microscope can be configured for simultaneous fluorescence and transmitted light-imaging techniques such as phase contrast and differential interference contrast (DIC) microscopy. The Optiphot-2 has an automated, computer-driven stage and focus system, which permits the user to store coordinates of objects and recall them at a later time. Nikon's high-transmission CF N Plan Fluor objectives have a high numerical aperture (a mathematical representation of the light-gathering capability of a lens), which helps create a brighter fluorescence image. The Axioplan-2 and Axiophot-2 digital microscopes from Carl Zeiss Inc. of Thornwood, N.Y., are multipurpose research microscopes equipped with Zeiss' ICS (Infinity Color-corrected System) optics. In particular, the Fluar ICS objective lenses, such as the Plan Neofluar, are high numerical aperture lenses designed to transmit very bright images over a wide spectral range. Filter cubes mounted on a reflector turret allow researchers to select up to five colors for individual or simultaneous fluorescence imaging. The Provis AX-70 research microscope from Olympus America Inc. of Mellville, N.Y., includes Olympus' advanced U.I.S. Infinity-corrected lenses for the creation of bright virtual images. The Provis is mounted on a Y-shaped stand, which provides easy access to control features. It offers up to five output ports for connecting still cameras, video equipment, or higher-sensitivity detection devices, such as a photomultiplier tube (PMT) or charge-coupled device (CCD) camera. The Leica DM LB microscope from Leica Inc. in Deerfield, Ill., is a general-purpose microscope suitable for many light microscopy applications. It can be equipped for fluorescence microscopy with the addition of a fluorescence illuminator, which features a turret in which up to four filter cubes can be mounted. With some confocal microscopes, images are formed a single point at a time. The high-energy, highly focused light that is used in confocal microscopy makes this technique ideal for applications involving fluorescent probes. Whereas a traditional fluorescence microscope outfitted with an epi-fluorescence illuminator reveals structures by collecting light coming from different depths in a specimen, a confocal microscope produces images by collecting light from a single focal plane. Compared with a light microscope, a confocal microscope improves axial resolution (the ability to discriminate between two close objects) by up to 30 percent. With confocal microscopy, a focused beam of light (typically from a laser) illuminates a single point in the specimen. Emitted fluorescent light returns through the same aperture before reaching a photodetector such as a PMT or cooled CCD camera. In this way, the out-of-focus light, which can create a blurry background "haze," is rejected. Point by point, the information is digitized and assembled into a three-dimensional reconstruction of the cell under study. Most laser scanning confocal microscopes consist of a special confocal unit attached to a conventional fluorescence microscope. The LSM 410 invert laser scan microscope from Zeiss, for instance, is an integration of confocal microscopy into Zeiss' Axiovert inverted light microscope. Many confocal microscopes are sold as a system that incorporates the confocal unit with a photodetection device (a PMT or digital camera), along with a computer and dedicated image-analysis software. The creation of confocal images generally requires digital manipulation to convert photons of light first into data points and finally into a visual re-creation of the specimen. As a result, most confocal images are viewed on a video or computer screen and not through the microscope's ocular lens. COMPREHENSIVE: Meridian Instruments' Ultima confocal laser system. The InSight Plus Laser Scanning Confocal Microscope system by Meridian Instruments Inc. of Okemos, Mich., is unique in that confocal images can be directly viewed, according to the company. "With the InSight systems, ocular viewing permits investigators to see optical sections in real time and real color," reports Peggy Wade, vice president for applications development at Meridian. The InSight Plus system, which can be used with microscopes from Olympus, Nikon, and Zeiss, is a modular system that provides users with flexibility. "The basic InSight Plus unit consists of the confocal scanner and controller only, and so researchers that already have some of the components do not have to purchase an entire system," Wade says. Options allow researchers to build an integrated system that offers computer-controlled imaging on PC or Macintosh computers of multiple fluorescence colors. In addition to the high speed and ocular viewing capabilities, the Sight Point system, Meridian's newest product, permits investigators to switch between previewing specimens through the ocular lenses to higher-resolution point scanning with a single keystroke, the company states. Dual lasers and a computer-controlled filter wheel allow for multicolor imaging. The system also enables ratiometric imaging, in which spectral shifts caused by a dye binding to its target ion are measured. An additional advantage of the InSight Point is precise control of each laser. With this feature, multiple probes are excited separately, so each color is bright and photobleaching (destruction of the dye) is minimized, according to Meridian. Meridian's Ultima system is a comprehensive laser confocal workstation that consists of a confocal imaging and data-collection system and three high-sensitivity PMTs, along with ultraviolet and visible lasers that provide simultaneous laser excitation at three wavelengths. With this system, operations such as simple optimization routines as well as complex 3-D reconstructions are controlled via a user-friendly interface, according to the company. This system offers stage-scanning capability, which permits high-resolution confocal scans of specimens larger than the field of view. The Sarastro 2000 CLSM and the MultiProbe 2010 Inverted CLSM systems from Molecular Dynamics of Sunnyvale, Calif., are integrated confocal laser scanning microscope systems. Molecular Dynamics' ImageSpace software, when used with these systems, performs the data-acquisition, image-processing, and data-quantification steps necessary for measuring and analyzing 3-D volumes. Using a laser as an illumination source has its inherent drawbacks, such as the possibility that the high-energy light will overheat the specimen in addition to illuminating it. Two-photon excitation is a way of avoiding this problem. In this technique, two lower-energy (infrared) photons from a titanium/sapphire laser are used in place of a single photon of higher-energy light to excite the dye. When two-photon excitation is used with a confocal microscope, the tissue above and below the plane of focus is subjected only to the lower-energy infrared light. This reduces the potential for heating up the specimen as well as the amount of photobleaching. Optical sectioning-creating images one focal plane at a time-may also be accomplished without a confocal microscope. Optical sectioning with a conventional microscope is a concept developed within the last 10 years by Fredric Fay, director of the biomedical imaging group at the University of Massachusetts Medical Center in Worcester, and by David Agard and John Sedat in the department of biochemistry and biophysics at the University of California, San Francisco. The technology is based on an algorithm that constructs the final image from a series of images collected in three dimensions. SEE IT IN 3-D: Applied Precision's DeltaVision system acquires images of fluorescently labeled specimens. The DeltaVision system from Applied Precision Inc. in Mercer Island, Wash., is a wide-field, optically sectioning microscope workstation designed to acquire three-dimensional images of fluorescently labeled specimens. The CELLscan system from Scanalytics/CSPI of Billerica, Mass., is also based on the concept of optical sectioning. Images are obtained at sequential optical depths and sent to an array-processing computer. The computer constructs a 3-D representation of the sample by considering the fluorescent signals obtained from various depths. To produce the high-resolution, sharply detailed images obtained with these systems, the computer reassigns the background "noise" to its source while analyzing the information gained at each focal plane. This computationally intense process has become commercially feasible only within the last five years, with the arrival on the microcomputer scene of the Pentium PC from Santa Clara, Calif.-based Intel Corp. and Cupertino, Calif.-based Apple Computer Inc.'s Power Macintosh. Some objects naturally emit fluorescent light, a phenomenon called autofluorescence. Other specimens, including most types of cells, must be stained with a fluorescent dye (fluorochrome) before they can be detected by fluorescence microscopy. Fluorochromes absorb light at a characteristic wavelength, and then re-emit lower-energy light at a longer wavelength. The fluorochrome fluorescein isothiocyanate (FITC), for example, absorbs light in the blue region of the spectrum at a wavelength of 490 nm. Electrons in the dye move to a higher energy level as a result. As the electrons fall to a lower energy level, the fluorochrome radiates green light at 525 nm, a longer-wavelength, lower-energy state. This change in color is referred to as a spectral shift. VERSATILE: Leica's DM LB can be adapted for fluroescence microscopy. FOR IMAGE PROCESSING: Molecular Dynamics' ImageSpace software Molecular Probes Inc. of Eugene, Ore., markets a wide selection of fluorescent dyes, including fura-2, calcium green, and fluo-3. Investigators may choose from among these according to the desired application and the type of fluorescent imaging device to be used. "Researchers need to consider the spectral properties of the dye and the type of microscope with which they work," comments Josh Stahl, technical assistance representative for Molecular Probes. "The various types of microscopes work better with different wavelengths of light. For example, for a confocal microscope equipped with an argon laser, the best results would be achieved with a fluorescent indicator that is excited at a wavelength of 480 nm." Using fluorescent dyes that specifically label a particular intracellular chemical, researchers can study the synchronous biochemical changes that occur as cells send and receive chemical messages. Calcium, for example, plays an important role in signaling for several types of cells. One can observe the movement of such intracellular indicators as calcium, sodium potassium, and hydrogen by adding fluorescent probes that specifically label the target chemical. "Once the dye binds to its intracellular target, a spectral shift occurs," explains Stahl. Thus, a dye that starts out blue may fluoresce yellow or green after binding to calcium. When a specimen is viewed with a fluorescence microscope, the color or intensity of the emitted light corresponds to the amount of free calcium in the cell. CONFOCAL SYSTEM: The 2010 CLSM unit from Molecular Dynamics While many fluorescent indicators undergo a spectral shift after binding to calcium, others, called fluorescence lifetime indicators, show a change in the length of the fluorochrome's excited state. "A fluorescence lifetime is essentially the time that elapses between when the fluorophore absorbs a photon of light and subsequently releases one of a different wavelength," explains William Mantulin, director of the Laboratory for Fluorescence Dynamics, a National Institutes of Health-sponsored research technology center at the University of Illinois, Urbana-Champaign. The lifetime of an excited fluorochrome lasts only nanoseconds and is generally quite predictable. When lifetime indicators, such as dye fluo-3, bind to their intracellular target, the change in the fluorescence lifetime leaves a characteristic signature behind for the observer to interpret. "Cells that fluoresce more brightly than other cells may contain more dye, or they might be thicker with a higher concentration of intracellular calcium," says Mantulin. "To resolve this ambiguity, we can measure the fluorescence lifetime of the dye." Fluorochromes hooked to molecules of DNA or to antibodies make powerful probes in biological systems. Oncor Inc., based in Gaithersburg, Md., offers biological probes for chromosomes, including FITC-labeled probes such as Chromasome Y a-Satellite, Chromasome 15 Classical Satellite, and Chromosome 7 Cocktail. When the probe binds to its DNA target sequence, investigators can identify the chromosome or chromosomal region by using a fluorescent microscope to observe the location of the brightly colored spots. This technique, called fluorescence in situ hybridization (FISH), is becoming popular as a means to study cellular genetics. "FISH permits very precise spatial resolution of morphological and genomic structures," says Mel Brenner, technical manager of the biomedical department at Nikon. "Depending on the probes used, the technique can be utilized to identify the entire genome of a particular species, individual chromosomes or chromosomal-specific regions, or single-copy unique DNA sequences found within the genome." The technique of immunofluorescence combines the specificity of antibodies with the sensitivity of fluorescence, creating a powerful detection system for biomolecules such as proteins and nucleic acids. Attaching a fluorescent dye to an antibody creates a colorful probe for proteins or other antigens that might be located intracellularly or on a cell's surface. Recently, for example, a team of researchers from Rockefeller University and Memorial Sloan-Kettering Cancer Center in New York City utilized both FISH and immunofluorescence techniques to localize in cells a factor identified as TRF (for telomeric repeat binding factor), a protein component of human telomeres (L. Chong et al., Science, 270:1663-7, 1995). Telomeres are elements found at the tips of chromosomes that apparently shield the ends from degradation or fusion. The loss of these repeated sequences may result in a form of genome instability that has been linked to cancer (R. Lewis, The Scientist, Feb. 19, 1996, page 12). By combining FISH with fluorescently labeled antibodies specific for the TRF factor, the investigators were able to pinpoint coinciding locations in cells for both telo-meric DNA and the TRF protein, leading them to conclude that TRF is a major telomeric protein. Their findings also raised the possibility that the harmful effects resulting from a loss of telomeres may be caused by failure of the chromosome ends to bind with protective telomeric proteins, including TRF. As important as fluorescence microscopy is to the field of cell biology, the technology is surprisingly absent from the undergraduate laboratory curriculum at colleges and universities in the United States. "Currently, fluorescence microscopy is rarely taught at the undergraduate level in a hands-on way," comments Intracellular Imaging's Gruenstein. A key reason for this may be the cost of the instrumentation. Whereas a light microscope for student use may be obtained for as little as $1,000 to $1,500, fluorescence microscopes can cost 20 times as much. To overcome problems of high cost, Intracellular Imaging has introduced the Groony Epifluorescence Module, which is a unit that converts a conventional light microscope into a fluorescence microscope. Although the module is suitable for research applications, the driving force behind the development of the unit was education, according to the company. For less than $2,000 per unit, colleges and universities are able to retrofit their existing bright-field microscopes with fluorescence optics. "In this way, undergraduate students will gain valuable hands-on experience in fluorescence microscopy techniques," states Gruenstein. Fluorescence microscopy techniques like intracellular ion imaging and confocal laser scanning microscopy have essentially revolutionized life scientists' ability to observe minute subcellular structures such as microtubules, as well as dynamic cellular events as they occur in living cells. If the current trends in microscopy continue, the skills of students versed in fluorescence microscopy will be in great demand. Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

With Fluorescence Microscopy, Researchers See Cells In A New Light

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Holly Ahern

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April 1996

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