News
ANALYTICAL CURRENTS
Imaging fluorometer determines photosynthetic function
potential for activity of the photosynthetic Vibrational spectra apparatus, the demands of the "dark reaction," the health of the leaf, and the plant dig out buried amino adaption to environmental conditions. Be- acids cause they change quickly and are evenly distributed across the leaf, however, these Buried within hydrophobic regions of profluorescence patterns are difficult to inteins such as thioredoxin and visual rhoScientists at four Western universities terpret. dopsin, aspartic and glutamic acids are have developed a new imaging fluorescence spectrophotometer that they beDaley and colleagues used a computer- known to be protonated even at pH values of 7 and higher. Under these condilieve will improve agricultural investigainterfaced imaging spectrophotometer tions, the carbonyl stretching frequencies tions by providing information on equipped with a charge-coupled device of aspartic and glutamic acids shift to photosynthetic leaf function. Growth of camera that can acquire spectra for higher frequencies. In some cases the agricultural crops depends on appropriate 31,680 positions per sample to image carbonyl stretch may appear above 1760 irrigation, fertilization, and exterminaleaves from several types of plants grown tion; and visualization of leaves by trained in a greenhouse, including coffee, nutmeg cm-1. However, little work has been done "scouts" is commonly used to plan spray, cedar, Amaranthus cruentus, and Digiirrigation, and fertilization schedules. The talis purpurea. Two simple filters were new system, developed by Li Ning and used to remove excitation emission overLarry S. Daley of Oregon State University, lap, and software based on a novel approxGerald E. Edwards of Washington State imation was used to image the timeUniversity, Garry A. Strobel of Montana dependent fluorescence of photosystem II State University, and James B. Callis of the across the surface of the leaf. University of Washington, is based on deThe new instrument has the potential tecting the changing patterns of fluoresfor research of plant pathology and ofcence that dark-adapted photosynthetic fers numerous applied uses, say the retissues generate immediately after illumisearchers. The simple design of the sysnation. tem, compared with previous designs, The strength and composition of these should facilitate commercial production. Laboratory studies have shown that the complex fluorescence signals in a given new technique can be used to determine plant can be related to the structure and the extent of freeze damage and the impact of FT-IR spectrum of the protein thioredoxin herbicides atpH 6.5 and room temperature showing a and to detect shifted carbonyl peak of a buried amino stress inacid (inset). duced in plants under to relate these shifting carbonyl stretching attack by frequencies for aspartic and glutamic acplant pathoids with the kinds of environments found gens. The au- inside such proteins. thors have Andrei K. Dioumaev and Mark S. Braialso reconfig- man of the University of Virginia Health ured the inSciences Center tackle this problem by strument to comparing carbonyl stretching frequenimage at a cies of several simple aliphatic carboxydistance of 7 lic acids with more complex systems, such m for potenas TV-acetylaspartic acid oc-amide, in sevtial remote eral different solvents. The authors resensing apport that propionic or butyric acid are plications. good models for aspartic and glutamic (Appl. Spec- acids in these types of proteins. In nonImage of an Amaranthus cruentus leaf 12 h after exposure to a solution trosc. 1995, hydrogen-bonding solvents, the carbonyl of DCMU, an herbicide that interrupts photosynthesis. (Adapted with 49,1381-89) stretching frequency for these acids permission of the Society for Applied Spectroscopy.) Analytical Chemistry News & Features, January 1, 1996 15 A
News varies linearly with Onsanger's parameter, a value proportional to the strength of the solute-induced electrostatic field of the solvent. Their studies indicate that the only way to model carbonyl frequencies above 1760 cm-1 is to describe the aspartic and glutamic acid residues as free of hydrogenbonding and to set the effective dielectric constant in the vicinity of the -COOH
group at only around 2. The authors predict that when this model is applied to the Asp-85 residue in bacteriorhodopsin in the unphotolyzed protein (vco =1730 cm-1), the amino acid is in a relatively polar environment that changes drastically to a highly nonpolar situation upon photoconversion to the M state (v co = 1762 cm"1). (J.Am. Chem. Soc. 1995,117,1057210574)
Measuring fluorescence decay with a confocal microscope
tion light is selected from the white synchrotron radiation spectrum with a bandpass filter matching the absorption band of the fluorophore, and decay curves of fluorescence intensity emitted by a preselected microvolume in the sample under investigation are acquired using standard time-correlated single-photon counting techniques. Unlike laser-based setups, the synchrotron radiation-based microscope can measure fluorescence decay of all molecules with absorption bands in the wavelength range of 250-700 nm. These lifetime measurements differ from conventional lifetime determinations on macroscopic samples in that both the excitation and the detection of fluorescence emissions are performed over a large solid angle, which obviates the need for defining the polarization directions of the excitation and emitted light beams. Use of the large solid angle also results in obtaining a high fluorescence emission collection efficiency (-25%). The first lifetime measurements performed on Syclops were of a coumestrol solution; subsequent experiments were performed on different cellular compartments of living cells incubated with coumestrol. Results from studies of coumestrol-stained H540 tumor Leydig cells indicate that fluorescence in the nucleus is ~ 40 times weaker than that from the cytoplasm, which agrees with results obtained from earlier work. The fluorescence decay curves, however, are virtually identical, an indication that the difference influorescenceis caused by inhomogeneous distribution of coumestrol in the cell rather than by dynamic quenching effects. Gerritsen and colleagues believe that the Syclops confocal microscope opens up applications for fluorescence lifetime measurements in selected microvolumes and that such applications are limited only by the absorption in the tissue layers between the probed volume and the sample surface. (Appl. Spectrosc. 1995, 49,1469-73)
In biological research, fluorescence techniques are widely used to selectively detect and image low levels of dye-labeled material. Quantitative fluorescence microscopy can be used to measure the local dye concentration, but accurate measurement is complicated by quenching from environmental factors such as pH, ion concentrations, and the presence of oxygen. The effects of these distortions can be quantitatively estimated by measuring the time-resolved decay of the fluorescence intensity; this has caused increased interest in analytical methods that can be used to measure fluorescent lifetimes in microscopic volumes. Using a new confocal microscope they term "Syclops," Hans C. Gerritsen and colleagues at Utrecht University (The Netherlands), SERC Laboratory Daresbury (U.K.), and Erasmus University (The Netherlands) have successfully measured fluorescence decay from a confocal spot with a volume of 3 um3. Syclops used the Doresbury Synchrotron Radiation Source as a pulsed light source. Visible or UV excita-
Fluorescence decays in the nucleus (upper trace) and the cytoplasm (lower trace) of a tumor Leydig cell. (Adapted with permission of the Society for Applied Spectroscopy.) 16 A
Analytical Chemistry News & Features, January 1, 1996
Chromatograms showing separations of various organometallic compounds using ICPMS (top) versus an FID output. Both are at optimum conditions, but the TMAs peak in the lower chromatogram coelutes with the solvent peak. (Adapted with permission of Preston Publications.)
Speciation using SFC/ICPMS Because the toxicity of metal species varies significantly, speciation information is required to fairly assess environmental and health risks. For example, monomethyl arsenate is about 1000-fold more toxic than arsenobetaine. In an effort to develop an analytical method that is more sensitive than LC coupled with atomic spectrometry, Joseph A. Caruso and co-workers at the University of Cincinnati explored the use of SFC coupled with detection by ICPMS for speciation of organometallic compounds. They analyzed stock solutions containing three arsenic, triphenyl antimony, and diphenyl mercury compounds and compared results with those obtained using flame ionization detection (FID). Using time-resolved acquisition software to monitor different elements, the researchers were able to determine allfivecompounds from a single injection by ICPMS. They find that switching solvents from methylene chloride to methanol reduces polyatomic interferences in the ICPMS spectrum from 40Ar35Cl+. Detection limits are 102-103 times lower than those for FID, falling into the picogram range. (J. Chromatogr. Sci. 1995, 33, 606-10)
