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the front-face configuration, the excitation source is in front of the sample. This setup allows another polarizer to be placed between the reference fluorophore and the sample. The additional polarizer selects the perpendicular component of the sample emission and extends the range of the compensation angle to 90°. At this stage, Lakowicz says there's no clear advantage for either method of polarizing the emission from the reference fluorophore, although the stretchedfilmis easier to construct. "But you might not have a perfectly matched color, because your fluorophore might not be the same color in the film," he says. "With the same self-reference, you have no color matching
problems, but it means there's probably another solution within the device." The sensor's reliance on the human eye to detect the intensity differences might seem to limit the accuracy. Not so, according to Lakowicz. "We knew it was quite accurate, but we didn't have a number for it. We went back and checked," he says. "The human eye is good to 10% relative accuracy. That 10% relative accuracy corresponds to one to two degrees of angular displacement. We further translated that into concentration ranges. In terms of pH, it is close to being good enough for clinical accuracy. But at this moment, it is not as accurate as highly standardized blood pH measurements."
Thus far, the sensors have been used to measure the concentration of Rhodamine B in intralipid, a highly scattering solution that mimics the scattering properties of skin, and to measure pH using 6-carboxyfluorescein. With compensation angles typically accurate to 1°, the pH values are accurate to ±0.1 pH unit. Lakowicz envisions the polarization sensors being used in a variety of settings. "The neat trick is that you're doing fluorescence sensing, which has numerous applications in biotechnology, DNA analysis, high-throughput screening, and drug discovery, with nothing more than a light source, your eyes, and a couple of polarizers." Celia Henry
NEWS FROM HPCE ’99 Britt Erickson reports from Palm Springs, CA.
Coordination ionspray MS Most nonpolar compounds are not amenable to electrospray ionization (ESI) because they lack a site for protonation or deprotonation. To allow such compounds to be characterized by MS, Ernst Bayer and co-workers at Universitat Tubingen (Germany) have developed a novel chemical ionization method, called coordination ionspray (CIS), which transforms the analytes to positively or negatively charged coordination compounds. In most cases the kinetics of complex formation are rapid, so postcolumn formation does not lead to band broadening when the technique is coupled with HPLC, CE, or capillary electrochromatography. Terpenes, neutral aromatic compounds, vitamins A and D, carotenoids, and unsaturated fatty acids are transformed on line via either pre- or postcolumn reaction and nebulized into the mass spectrometer as palladium (II) or silver© complexes, sugars and saccharides as boron complexes, and neutral polypeptides as lithium complexes. In tandem MS mode, coordinated daughter-ion fr3.|?ments are often obtained providing additional structural information (e.g. Ag+-coordinated fragments of unsaturated fatty acids allow the assignment of the position of the double bond) In contrast to ESI, CIS does not require strong electric fields or the presence of sites that can be protonated or deprotonated. In addition, CIS-MS offers sensitivity in the 244 A
Olefins analyzed by CIS-MS.
lower femtomole region. CIS-MS combined with a separation technique such as HPLC or CE opens up new possibilities for the characterization of a wide variety of neutral or weakly polar compounds, which cannot be subjected to ESI-MS.
Correlating enzymatic activity and cell cycle Single-cell analysis offers a means for detecting variations from one cell to the next within a population of cells. Such variations arise largely from cells being at different stages in the cell cycle. In contrast to normal cells, cancer cells proliferate rapidly. Because cancer cells progress through the cell cycle asynchronously, the enzymatic activity of tumor cells varies significantlyfromcell to cell. Classic cytometry techniques, such as flow cytometry and fluorescent image cytometry, have been used to study variations in enzymatic activities associated with different phases of the cell cycle; however, only a limited number of enzymes (rarely more than
Analytical Chemistry News & Features, April 1, 1999
three) can be assayed simultaneously. Sergey Krylov, Norman Dovichi, and coworkers at the University of Alberta (Canada) have developed an approach for assaying multiple enzymatic activities in individual cells by combining CE and fluorescent image microscopy. To monitor enzymatic activities, cells are incubated with afluorescentlylabeled metabolic probe. The probe is taken up by the cells and converted by several enzymes to metabolic products, which retain the fluorescent label. A single cell is injected into the capillary and lysed. The products are then separated by CE and detected by laser-inducedfluorescence.Knowing the concentration of the metabolic products allows for the indirect assessment of the enzymatic activities in individual cells. The cell's phase in the cell cycle is determined by measuring the amount of DNA in the cell before it is injected into the capillary. Cellular DNA content varies according to the phase in the cell cycle—in the Gl phase, cells contain one set of chromosomes; in the S phase, DNA content increases dramatically as a result of DNA replication; and in the G2 and M phases, cells contain two complete sets of chromosomes. Hoescht 33342—a cell-permeable, DNA-intercalating fluorescent dye—is used to stain DNA proportionally. Hoescht fluorescencefroma single cell is measured with a photomultiplier tube detector mounted on afluorescentmicroscope. By measuring the amount of DNA in individual cells they C3.n be ciscribed to one of three phases Gl S or G2/M The researchers use the technique to
