Dynamics Author: Howard Goldner During the past few decades, fluorescence spectroscopy has developed into an integral technique in many scientific disciplines. In the life sciences, it is implemented extensively in areas such as biochemistry, biophysics, and cell biology for a variety of applications, ranging from basic assay-related quantitative measurements to DNA sequencing.
Locates Molecules:Fluorolog-t2 Spectrometer
More recently, advances in instrumentation, laser technology, and fluorescent dyes (or probes) are aiding researchers in their efforts to better understand the dynamics of complex biomaterials such as proteins, membranes, and nucleic acids. Fluorescence- microscopy methods, for example, are increasingly being used to study the localization and movement of intracellular substances (H. Ahern, The Scientist, April 17, 1995, page 18).
"Scientists like to use fluorescence spectroscopy for two reasons," explains William Mantulin, an adjunct associate professor of biochemistry and biophysics and the director of the Laboratory for Fluorescence Dynamics, a National Institutes of Health-sponsored research resource center at the University of Illinois. "First of all, the technology has very high sensitivity, allowing many applications for analytical measurements. But also, it can provide important dynamic information about the environment in biosystems."
Mantulin's laboratory is primarily a research and development center for the advancement of fluorescence technologies and applications. However, the facility also provides a service function by offering a suitable environment for non-NIH researchers who are interested in using fluorescence to conduct their own experiments. It's a good way to try out the system without incurring major capital expenses, according to Mantulin.
Typically aromatic, fluorescence molecules display light absorption from the ultraviolet through the visible regions of the spectrum. Tryptophan and tyrosine in proteins are examples of common, naturally occurring fluorescent substances.
When a molecule absorbs energy as light, it goes from the ground state to an excited state, and then releases excess energy via various pathways, including fluorescence emission. The delay between the initial absorption and the emission is referred to as the fluorescence lifetime, and occurs in nanoseconds.
During this very brief period, the excited molecule is often modified through interaction with other molecules in its vicinity. By measuring certain parameters of the emitted light, including time, wavelength, and intensity, researchers can obtain a "fingerprint" of the molecule's environment.
The major advantage of this technique is that many biological lifetimes fall in the 1- to 20-nanosecond range, and therefore coincide almost perfectly with this time scale. "Fluorescence spectroscopy is one of the few techniques that provides the time resolution to study protein properties on the nanosecond scale," says Enrico Gratton, a professor of physics and principal investigator of the Laboratory for Fluorescence Dynamics at the University of Illinois. In a sense, lifetime-based fluorescence can function as a molecular stopwatch to record events in nanoseconds with high sensitivity and selectivity. Many researchers are currently using this technique to study the dynamics of folding, ligand-binding, and substrate-binding reactions of proteins, and to monitor the compositional changes of membranes. "We are looking at protein conformation and protein-ligand interactions using fluorescence instrumentation from Photon Technology International [South Brunswick, N.J.]," reports Robert Copeland, a principal research scientist at DuPont Merck Pharmaceutical Co. in Wilmington, Del.
Fluorescence resonance energy transfer (FRET) is a mechanism whereby the excited-state energy is transferred from an energy donor to an acceptor to determine distances ranging from about 15 to 75 angstroms between sites on macromolecules.
Distances in this range are generally comparable to the thickness of membranes, or the diameter of many proteins, and are often very difficult to measure by other techniques, notes Gratton. Although X-ray analysis would be a possibility if substances could be formed into crystals, most materials in the life sciences--such as membranes, protein complexes, and nucleic acids--cannot be crystallized. Therefore, "fluorescence energy transfer is one of the few methods currently available that yields important information about the relative positions of different parts of a molecule," he explains.
Mantulin agrees that FRET is "a good way to get ideas about spatial distribution between regions where you don't have any crystal-structure information, such as binding a protein to a large DNA molecule." He also describes another process, known as fluorescence quenching, that provides information about the dynamics of a biosystem in cases in which structural information is not available.
A wide variety of small molecules or ions, such as iodide, oxygen, and acrylamide, act as fluorescence quenchers by decreasing the emission intensity of a material, explains Mantulin. Successive additions of a quencher will characterize the dynamics of the quenching process, and assist in determining the conformational changes when a reaction occurs, such as binding a substrate to an enzyme.
"We use fluorescence-quenching technology to analyze biological membrane structures," says Irwin London, a biochemistry professor at the State University of New York, Stony Brook. London makes model membranes with quenchers attached to the lipids at different depths and locations. He tries to determine the location of a fluorescent molecule from the amount of quenching using a Fluorolog-t2 spectrofluorometer from ISA JY-SPEX Inc., located in Edison, N.J.
London and his coworkers have developed a method allowing them to triangulate the position of the fluorescent molecule at the angstrom level. "At that high level of resolution, we're really getting a picture of where things are that can't be obtained by other techniques," he notes.
If a sample is illuminated with polarized light, the emission will also be polarized. This fluorescence-polarization phenomenon allows researchers to measure the rotational motions of proteins, and eventually determine molecular weights, because larger proteins rotate more slowly. Other applications include detecting the binding of one protein to another, or the self-association of a biomolecule into a dimer or trimer.
While doing postdoctoral research, Rolf Brandes, now a research assistant professor at the Loyola Medical School, located in Maywood, Ill., used a lifetime-fluorescence spectrometer from Rochester, N.Y.-based SLM-AMINCO to measure the fluorescence emitted from a beating heart.
"Using a fiber-optic cable connected to the excitation source, I radiated a whole heart and measured the calcium released as a function of flow to the coronary vessels," he says. That technique has also been performed in situ, by other researchers, using fiber-optic cables to measure fluorescence directly on the heart, according to Brandes.
In contrast to the lifetime-based, or time-resolved, fluorescence techniques, which tend to be more research-related, the more familiar steady-state mode refers to the measurement of fluorescence intensities while samples are continuously illuminated. Several instruments, including microplate readers and gel readers, are commercially available to measure fluorescence emissions at specific wavelengths for a variety of assays.
Included among these systems are the RF-1501 Spectrofluorophotometer from Shimadzu Scientific Instruments Inc. of Columbia, Md.; the Model LS-30 Luminescence Spectrometer from Norwalk, Conn.-based Perkin-Elmer Corp.; the Model TD-700 Laboratory Fluorometer from Turner Designs Inc., located in Sunnyvale, Calif.; and the FL500 Fluorescence Microplate Reader from Bio-Tek Instruments Inc. in Highland Park, Winooski, Vt.
Optical Technology Devices Inc., located in Elmsford, N.Y., has several manual and scanning spectrofluorometers available with a variety of features. And Stamford, Conn.-based Instrument Development Inc. offers the Laser-Trak-3000, an imaging system that uses laser-induced fluorescence to detect and quantitate DNA, RNA, and proteins separated by gel electrophoresis.
Also utilizing fluorescence as a method for detection is flow cytometry, a technique that rapidly counts and sorts heterogeneous suspensions of small particles or cells. "Not only can the fluorescence intensity of these cells be measured at several thousand particles per second, it's also possible to physically separate them out to purify a population," says Gary Durack, the director of the Biotechnology Flow Cytometry Center at the University of Illinois.
In addition, researchers can obtain information on several parameters simultaneously by attaching numerous fluorophores to the same cell, and measuring the emissions at specific wavelengths, he comments.
Among the flow cytometry systems that have been introduced recently are the EPICS Elite ESP instrument from Miami-based Coulter Corp. and the FACSCalibur from Becton Dickinson Immunocytometry Systems of San Jose, Calif.
Many biological substances are fluorescent in their natural state, and can be used as indicators without disturbing the system in any way. However, if there are no natural markers, an extrinsic fluorophore, or probe, may be introduced to reveal the structure and function of macromolecules.
"The development of fluorescent probes has enabled researchers to identify specific components or functions of living cells," says Iain Johnson, a technical communications specialist at Molecular Probes Inc., a supplier of fluorescent indicators located in Eugene, Ore. "Even when molecules in nature are fluorescent, the fluorescence signals are often not sufficiently specific to characterize a particular material in the presence of many others," explains Johnson.
The strategy around this problem is to isolate the material of interest by labeling it with a fluorescent dye that is extrinsic to the system. A particular component can thereby be identified from a complex mixture of all the other molecules in a biological system. "A constant design challenge [for manufacturers] is to develop fluorescent probes that are detectable at regions of the spectrum where the background is low, or to amplify the fluorescence signal so that its level is high relative to the background," says Johnson.
With immunofluorescence applications, for example, the fluorescent probe is a labeled antibody. To detect the presence of an antigen, often a fixed cell, a fluorescent dye is attached to an antibody. In situations in which certain antibodies cannot be labeled, a secondary antibody can be used as the probe to the first antibody, Johnson points out: "It's like building layers on a sandwich. Weak signals can be amplified in this way, by building layers one on top of another."
Lanthanide chelation, a technique commercialized as part of the DELPHIA Research System by Wallac Inc.--a Turku, Finland-based company with U.S. facilities located in Gaithersburg, Md.--offers another approach to fluorescent labeling.
"We use the lanthanide elements, especially europium, terbium, samarium, and dysprosium, to form highly fluorescent chelate labels," says Ann Christine Sundell, a Wallac product specialist. The system's fluorescence lifetimes are considerably longer than most probes, making it possible to measure their emissions well after the background interference has decayed, she claims.
Sensitivity is further enhanced because the lanthanides exhibit sharp emission peaks at specific wavelengths. In addition, it's possible to perform multi-label assays for the detection of more than one parameter on the same sample. Applications of this technique include DNA hybridization, numerous immunoassays, and the quantification of polymerase chain reaction products.
Fluorimetric enzyme determinations have also become popular, owing to the speed of the assay and the sensitivity of the method. As a result, "fluorogenic enzyme substrates have found widespread applications, especially, in microbiology, for the detection and identification of bacteria; in molecular biology, for detecting reporter gene expression; and in the areas of analytical cytometry, immunofluorimetry, and clinical analysis," reports Ernst Koller, a consultant for Lamda Fluorescence Co., located in Pleasant Gap, Pa.
In addition, chemists in Japan recently reported (T.D. James, K.R.A. Samankumara Sandanayake, S. Shinkal, Nature, 374:345-7, 1995) the design of a fluorescent molecular sensor that distinguishes between optical isomers of monosaccharides. This new development is expected to be of particular importance to the areas of drug design and synthesis, in which there is increasing regulatory pressure in the United States to produce enantiomerically pure products. Trends In Fluorescence
From a marketing standpoint, Jim Mattheis, fluorescence product specialist at ISA JY-SPEX, sees a better-than-average growth rate in high-performance instruments. "We're taking advantage of advances in materials and electronics to make smaller footprint [space-saving] instruments at lower cost," Mattheis notes.
And David Smith, marketing services manager at Photon Technology, claims his company is "poised and ready for any sudden expansion" in one of the specific technique-related areas. "Because we take a modular approach with our hardware, we can produce new configurations of fluorescence instruments within months rather than years."
Vijai Tyagi, marketing manager at ISS Inc., a Champaign, Ill.- based manufacturer of fluorescence instrumentation, sees "a definite trend in the use of fluorescence as a replacement for radiolabeled materials that trace the path of certain compounds inside cells or within the body." This direction results not only from the high sensitivity of fluorescence detection, but also from researchers' desire to eliminate the use of radioisotopes, with their disposal concerns and limited lifetimes, from the laboratory and clinical areas.
Although lifetime-fluorescence techniques have been primarily a research tool in biochemistry and biophysics, medical testing based on this technique appears to be rapidly migrating to the clinical laboratory, to the doctor's office, and even to home health care.
"Because of major changes in technology, I see time-resolved fluorescence moving very quickly into the clinical world," says Joseph Lakowicz, director of the Center for Fluorescence Spectroscopy at the University of Maryland School of Medicine. Heading a facility similar to the laboratory at the University of Illinois, Lakowicz is "pushing the boundaries of fluorescence applications."
He explains: "There is a growing recognition in the value of using longer-wavelength probes because tissues are nonabsorbing at these wavelengths." He predicts that it may be possible to perform noninvasive diagnostics, such as determining the tissue levels of glucose, by using probe implants that can be excited with simple, hand-held laser diode sources, similar to those used in compact disc players.
(The Scientist, Vol:9, #12, pg.18, June 12, 1995)
(Copyright, The Scientist, Inc.)