Fluorescence Spectroscopy Shedding Light On Complex Biosystems

Complex Biosystems Spectroscopic detection of fluorescent molecules, either those found naturally or those colored with fluorescent dyes, has created new research possibilities for scientists who study proteins and DNA. With photonic instruments like spectrophotometers and spectrofluorimeters, life scientists can measure the low-intensity light emitted from fluorescent molecules. "Because the character of the light emitted from a molecule changes in respect to the environment the molecule is in, fluorescence is a very sensitive reporter of the conditions in the sample," says Richard DeSa, president and director of R&D at On-Line Instrument Systems Inc., a Bogart, Ga.-based company that specializes in spectrophotometric and spectrofluorimetric instruments with scanning capabilities. He adds that the fluorescence provides investigators with dynamic information about complex biosystems. Fluorescence techniques make it easier for life scientists to study the structure or dynamics of biomolecules, such as protein folding or how various molecules interact. Additionally, fluorescence spectroscopy enables researchers to make quantitative measurements of assays, to detect fluorescently stained DNA and proteins in gels, and to sequence DNA by semi- or fully automated techniques. Recent advances in laser technology, instrumentation, and, particularly, the development of fluorescent chemicals used to identify various cellular targets have made it easier for scientists to apply fluorescence spectroscopy in their research. "There is growing interest in fluorescence assays, which is being driven in part by the companies that develop and supply fluorescent probes," explains Ted Quigly, product manager at Bio-Tek Instruments Inc. of Winsooki, Vt. SPECTROFLUORIMETER: Bio-Tek Instruments FL500 Fluroescence Reader for fluorescenct microplate assays. Bio-Tek Instruments manufactures the FL500 Fluorescence Reader, a spectrofluorimeter used for reading fluorescent microplate assays. This automated instrument is capable of reading microplates containing six to 384 wells. Quigly notes that fluorescence assays that can be performed in microplates are being developed at an increasing rate. Molecular Probes Inc. of Eugene, Ore., supplies several fluorescent dyes that act as indicators in such assays. "Quantitative assays that can be performed in microplates include tests for single- and double-stranded DNA, for proteins, and also for particular types of cells," such as live cells vs. dead cells, he says. Even in situ assays, which usually are detected by microscopy, can be performed in larger-well microplates. Instruments that detect fluorescence in microplates or gels are based on two standard methods of measuring fluorescence: intensity and time. In the intensity-based, or steady-state, mode, specimens are continuously illuminated with light of a certain wavelength, while the intensity of the emitted light is measured. The LS-30 Luminescence Spectrometer from Perkin-Elmer Corp., based in Norwalk, Conn., and the RF-1501 Spectrofluorophotometer from Shimadzu Scientific Instruments Inc. of Columbia, Md., both are designed to measure fluorescence in biological assays performed in microplate wells. Several manual and scanning spectro-fluorimeters enable researchers to sense fluorescently stained samples in gels. Scanning spectrofluorimeters use laser beams to induce fluorescence. Stamford, Conn.-based Instrument Development Inc. offers the Laser Trak 3000, an imaging system that detects fluorescent molecules in gels. These molecules could be fragments of DNA generated by sequencing reactions or proteins labeled with a fluorescently tagged antibody. Instruments such as the Laser Trak generally allow investigators to measure fluorescence emissions at specific wavelengths, depending on the type of fluorochrome chosen. Instead of labeling samples with radioactivity and detecting them by autoradiography, fluorescence techniques permit sensitive detection of samples directly in gels. Additional benefits are less exposure to hazardous radioisotopes for the user and a much lower cost of disposal. CREATES IMAGES: The Fluorimager SI from Molecular Dynamics scans samples. The FluorImager SI from Molecular Dynamics Inc. of Sunnyvale, Calif., creates images of DNA and proteins in gels, in blots, on thin-layer chromatography plates, and in microplates. The optical system of the FluorImager SI focuses a scanning laser beam on the samples in a gel or a plate, which results in fluorescence, termed laser-induced fluorescence (LIF). The emitted light then is measured to produce a computerized image of the scanned sample. 'LIF' LINE: The P/ACE DNA laser-induced fluorescence (LIF) system from Beckman Instruments, Inc. The P/ACE DNA system from Beckman Instruments Inc. in Fullerton, Calif., is an automated system that separates pieces of DNA by capillary electrophoresis. An integrated LIF detector permits a researcher to analyze the separated fragment, which may consist of oligonucleotides, DNA amplified by the polymerase chain reaction (PCR), and restriction fragments. The ABI Prism 310 Genetic Analyzer from Perkin-Elmer's Applied Biosystems Division in Fullerton, Calif., also allows DNA fragments to be separated by capillary electrophoresis and analyzed with an LIF detector. ABI's LIF detector can sense four different fluorescent colors. Since each nucleotide in DNA will appear as a distinct color, all four sequencing reactions can be run in a single capillary tube. Many automated sequencing systems, such as the ALF DNA Analysis System and the ALFexpress System, both from Pharmacia Biotech in Piscataway, N.J., use LIF detectors to read sequences from slab gels, which are thin slabs of polyacrylamide molded between glass plates. With Perkin Elmer-Applied Biosystems' ABI Prism 377 DNA sequencer, DNA sequences are read from slab gels containing fluorescent fragments coded in four colors, as with the Prism 310 system. When the desired target is a cell rather than a molecule, flow cytometry may be a better tool. This technology permits investigators to rapidly identify, count, and even sort cells into homogeneous populations. In flow cytometry, particles or cells are first labeled with fluorochrome-tagged antibodies or other fluorescent tracers such as propidium iodide, specific for DNA. Once labeled, cells are placed in the flow cytometer, which provides a focused beam of laser light to excite the fluorochrome. The laser studies each cell as it passes single-file through a flow cell, at rates of up to thousands per second. A computer then electronically selects specific cells for analysis, or to separate them into a homogeneous cell population. FLOW CYTOMETRY SYSTEM: FACSCalibur equipment from Becton Dickinson The FACSCalibur system is a flow cytometry system available from Becton Dickinson Immunocytometry Systems of San Jose, Calif. Miami-based Coulter Corp. manufactures the EPICS Elite ESP, a flow cytometry system that now comes with software for the Windows environment. These systems are appropriate for cell sorting and other types of cellular analysis, including immunophenotyping and DNA analysis. For applications typically performed in medical laboratories, Coulter's EPICS XL system has been equipped with new software that allows the instrument to sense cells labeled with up to four fluorescent colors. Multicolor labeling allows researchers to study several cellular features at once by attaching different fluorochromes to the same cell and measuring the light emitted at specific wavelengths. An area in which fluorescence-based flow cytometry may have an impact in the future is FISH, or fluorescence in situ hybridization. FISH techniques use fluorescently labeled nucleic acid probes, which bind to similar target sequences directly in specimens mounted on slides or the surfaces of microplate wells. "Although originally intended to be [detected] by fluorescence microscopy, FISH techniques will be eventually adaptable to analysis by flow cytometry," predicts Roger Hubbard, director of Molecular Pathology Laboratory, a reference lab in Maryville, Tenn., that performs a wide range of DNA probe assays. FISH by flow cytometry-known as fluorescence in situ hybridization en solution, or FISHES-may eventually be applied to studies of gene expression at the level of individual cells. As opposed to intensity-based determinations of fluorescence, time-resolved spectroscopy measures changes in fluorescence emissions by a molecule over time. In time-resolved methods of fluorescence spectroscopy, samples are briefly illuminated and the length of the molecule's excited state is measured by the instrument. The excited state is the period between the initial absorption of light by the molecule and the subsequent fluorescence emission as the electrons return to a lower energy state. This period is referred to as fluorescence lifetime and lasts only nanoseconds. During a fluorochrome's brief life, the molecule may be modified through interactions with nearby molecules. For instance, the fluorescent dye calcium green undergoes a change in its lifetime when the chemical binds to calcium. By taking measurements-such as wavelength, intensity, and time-of the emitted light, researchers obtain a wealth of information about the environment in a particular biosystem. Selected Manufacturers of Fluorescence Spectroscopy Equipment and Supplies Applied Biosystems Division Perkin Elmer Corporation Beckman Instruments Inc. Becton Dickinson Immunocytometry Systems Bio-Tek Instruments Inc. Coulter Corporation Instrument Development Inc. ISA JY-SPEX Inc. ISS Inc. Meridian Instruments Inc. Molecular Dynamics Inc. Molecular Probes Inc. Nikon Inc. Olympus America Inc. On-line Instrument Systems Inc. Perkin-Elmer Corporation Pharmacia Biotech Photon Technology International Shimadzu Scientific Instruments Inc. Wallac Inc. Carl Zeiss Inc. Because this technique provides a nanosecond of information, time-resolved fluorescence spectroscopy is being applied to study the dynamics of protein folding, protein-ligand interactions, and substrate binding-events that occur on the same time scale. A technique known as fluorescence resonance energy transfer (FRET), in which excited-state energy is transferred from a donor molecule to an acceptor, permits researchers to determine spatial relationships between sites in proximity to each other, within a biological membrane, for example. Instruments for analytical fluorescence spectroscopy are equipped mostly with lasers, which provide a high-energy, cohesive source of illumination, and sensitive detectors such as cooled charge-coupled device (CCD) imagers. Many are linked with computers and powerful software necessary to make sense of the vast quantities of information output by the instruments. Spectrofluorimetry systems that allow researchers to measure the parameters of fluorescence lifetime also permit them to perform fluorescence quenching, in which certain molecules act to decrease the energy emitted by an excited fluorochrome. Such systems include the Fluorolog-t2, a spectrofluorimeter manufactured by ISA JY-SPEX Inc. in Edison, N.J. Other spectrofluorimeters are the TimeMaster series from Photon Technology International, located in South Brunswick, N.J., and the K2 from Champaign, Ill.-based ISS Inc. The DELPHIA Research System from Wallac Inc., a Finland-based company with U.S. headquarters in Gaithersburg, Md., is based on a labeling technique known as lanthanide chelation. With the DELPHIA system, the fluorescence lifetimes of various lanthanide probes are measured by a time-resolved fluorimeter. An advantage of the lanthanide fluorophores is their long life span, which makes it possible to measure emissions well after any background fluorescence has faded away. Each of the lanthanide elements exhibits a characteristic emission peak at specific wavelengths, which enhances the sensitivity of assays performed with this system. Each peak can be distinguished easily, which makes it possible for researchers to perform assays using more than one label. Applications for this technique include DNA hybridization, numerous immunoassays, and quantification of PCR products. MICROSCOPE SPECTROSCOPY: The Attofluor Digital Fluorescence Analyzer from Carl Zeiss, Inc. Other types of quantitative fluorescence spectroscopy may be performed using a microscope. "Modern fluorescence indicators are being attached to specific biochemical materials to provide detection, quantification, and evaluation of many biomedical structures and processes on a subcellular or cellular level," comments Thomas Calahan, senior application specialist for microscopy at Carl Zeiss Inc. in Thornwood, N.Y. "Microscope spectroscopy is a very important tool for studying minute amounts of both living and nonliving materials with extreme sensitivity." Zeiss offers several microscope systems, including the MPM Microscope Photometer Systems, the Confocor Fluorescence Correlation Spectrometer, the Attofluor Digital Fluorescence Analyzer, and the Confocal Laser Scan Mi-croscope, for quantitative fluorescence emission spectroscopy.When equipped with an appropriate photodetector and software system, research-grade fluorescence microscopes such as the Optiphot-2 from Nikon Inc. in Mellville, N.Y., and the Olympus IX-70 from Olympus America Inc. in San Jose, Calif., also can be used for spectroscopic determinations of microscopic samples. DUAL USE: Meridian's Ultima is a laser scanning confocal microscope system tha can be used for microscope spectroscopy. The Ultima is a laser scanning confocal microscope system from Meridian Instruments Inc. of Okemos, Mich., that can be used for microscope spectroscopy, as well. In addition to imaging, this instrument will generate several quantitation parameters, including fluorescence intensity and the number of stained particles within a cell. Using optional software, researchers can use the Ultima system to perform advanced applications such as adherent cell sorting, fluorescence redistribution after photobleaching, and monitoring of cell-to-cell communication. With microscope systems capable of fluorescence spectroscopy, researchers can determine ultraviolet and visible light (UV/VIS) absorption spectrum, make kinetic measurements, and perform fluorescence correlation spectroscopy on samples less than 1 micron in size. Other quantitative techniques, primarily for industrial applications, in which a microscope may be used are micro FT-IR spectroscopy and Raman spectroscopy, both analytical spectroscopic techniques to determine a compound's chemical composition. A relatively recent development in fluorescence spectroscopy is the OLIS RSM 1000F, developed by On-Line Instrument Systems. The OLIS RSM is a rapid-scanning spectrofluorimeter that can acquire emission data on a millisecond time scale. "The OLIS RSM is an instrument that falls in between the conventional fixed-wavelength fluorimeters and the faster instruments that measure fluorescence lifetimes in nanoseconds," explains Julie Ann Lorenz, vice president for marketing and communications at the company. The rapid-scanning OLIS instrument has a monochromator with the ability to scan a spectral region in one millisecond. Thus, spectral information can be obtained even for samples that are undergoing dynamic change, such as proteins folding and unfolding in response to changes in pH. "The OLIS instrument is very useful for making determinations as a function of time," says Sarah Green, an assistant professor of chemistry at Michigan Technological University in Houghton, Mich. "Changes that occur quickly, such as a ligand binding to copper, can be detected during the milliseconds that it took for the event to take place." "Our instrument is to a typical spectrofluorimeter as a video camera is to a still camera," maintains DeSa. "If you induce a change in a system and take pictures of it with a still camera, you get nothing but a blur. However, if you were to use a video camera, the changes could be clearly recorded." Fluorescence spectroscopy performed on either the microscopic scale or the macroscopic scale is providing researchers in biology and medicine with a highly sensitive means for studying the structure and dynamics of biological molecules. "Chemical imaging, which is a combination of spectroscopy and imaging techniques, provides data that contains both structural and compositional information, allowing samples to be probed, even dynamically, with unprecedented analytical power," comments Patrick Treado, an assistant professor in the department of chemistry at the University of Pittsburgh. Concludes Joseph Lakowicz, director of the Center for Fluorescence Spectroscopy at the University of Maryland School of Medicine in Baltimore: "There is no doubt that fluorescence spectroscopy methods have contributed immensely to our present understanding of biological molecules and their assemblies." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Fluorescence Spectroscopy Shedding Light On Complex Biosystems

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