The enormous advantage of fluorescence is that many lifetimes fall in the 1-to-20-nsec range. This time scale coincides almost perfectly with the time scale of molecular interactions in biological systems under physiologically active conditions. “Time-resolved” fluorescence methods use sophisticated hardware and methodology to resolve events that occur on this time scale.
In contrast, “steady-state” measurements are accomplished more simply. A continuous beam of light serves as an excitation source, and the resulting fluorescence is observed on a time scale appropriate for the experiment–generally on the order of milliseconds to seconds.
The information from steady-state and time-resolved measurements is complementary. However, in many applications, steady-state measurements may suffice for the process, or may be the only practical choice available.
“We use time-resolved and steady-state fluorescence spectroscopy in a variety of ways for drug research,” says Edmund Matayoshi, a biophysicist at Abbott Laboratories, Abbott Park, IL. “Fluorescence is a versatile and practical technique for studying the structural and functional properties of macromolecular drug targets, as well as for the development of screening assays that are crucial to the process of discovering new drugs.”
Recently, Matayoshi and co-workers developed an assay for the pro-tease enzyme encoded by human immunodeficiency virus 1 (HIV-1). This enzyme is a target for the design of selective AIDS therapeutics because it is essential to the growth of infectious virus particles.
At the National Institute of Mental Health, Bethesda, MD, Christian Felder is developing chemical probes that change their fluorescent properties as they bind to key molecules and ions in living cells.
“These sensitive biochemical detectors can be loaded into the interior or bound to the exterior of cells without altering the cells’ normal function,” says biochemist Felder. “Single cells containing the probes are then placed in a microscope that has been connected to a spectrofluorometer with a fiber optic bundle. The cells are illuminated at specific wavelengths, and their response to changes in calcium concentration is recorded with a CCD video camera, which outputs to a computer for analysis and storage.”
At present, the most widely used chemical probe is FURA-2, which changes its fluorescent emission as it binds to free calcium ions. Calcium is an important ion found in every cell of our body and is essential for normal growth and development.
“Calcium ions move in a distinct wavelike pattern within the cell, and both the amplitude as well as the frequency of calcium wave propagation may code important information to the cell,” says Felder.
Other probes have been developed that are sensitive to biologically important ions such as potassium, sodium, and magnesium. This new technology could lead to a noninvasive method of observing living cells.
Coupled with fiber optics and selective probes, fluorescence spectroscopy is emerging as a worthwhile technique for many new applications. Its future is indeed radiant.
Undersea Fluorescence Monitoring Helps Track Climate Changes
The measurement of carbon dioxide in the environment is of great current interest because of the concern that an increase in the C|O.sub.2~ level due to human activity may dramatically change the climate of the Earth.
Although computer models have substantiated this concern, a lack of data exists to validate the models because of the difficulty and expense of collecting and analyzing discrete water samples at various spots in the ocean.
In response to this need, David Walt, a chemist at Tufts Univ., Medford, MA, has developed an immersible fiber optic pH sensor that demonstrates the required sensitivity and precision for these undersea measurements.
Another useful technology was developed by Richard Thompson at the Univ. of Maryland Medical School, Baltimore. He invented an on-board fiber optic system to measure zinc and other nutrient cations in the ocean.
“In some geographic areas, the biomass productivity, such as plankton, appears to be limited by the concentration levels of these divalent ions,” says biochemist Thompson. “We needed a technique that provides sensitivity, speed, and selectivity, in addition to real-time monitoring. Fluorescence, coupled with fiber optics, has proven to be suitable for our use.”
Thompson’s sensor uses a metal-loenzyme as a sensing transducer to maximize sensitivity, which is necessary in complex media such as seawater and blood serum. Conventional metallofluorescent indicators are insufficiently selective for these applications.