News
Quantitative singlemolecule detection One strategy for single-molecule detection involves an "open detection volume element", in which a confocal optical system, rather than a physical cell, defines the detection volume. Claus A. M. Seidel and co-workers at Max-PlanckInstitut fur Biophysikalische Chemie (Germany) identify and quantify molecules in dilute solution with a confocal fluorescence microscope and a spectroscopic method called BIFL (burst integrated fluorescence lifetime) Two different types of information are recorded in BIFL—the time lag between photons and the arrival of a signal photon relative to the exciting laser pulse. These two pieces of information can be used to identify photon bursts and to calculate the fluorescence lifetime. Fluorescence can be distinguished from background by the time lag between consecutive photons, which is large for background pho-
Understanding tryptophan fluorescence The amino acid tryptophan is a popular probe for protein studies. Provided that the protein structure is known, detailed knowledge of the effects the surroundings have on tryptophan fluorescence and its quenching could make the amino acid a probe for conformation changes and interaction with other molecules with atomic resolution. Mary D. Barkley and Yu Chen of Case Western Reserve University have taken a step toward that understanding by unraveling some of the contributions to tryptophan fluorescence quenching. They determined the amino acid side chains that quench the fluorescence of the indole group)—tryptophan's fluorescent side chain—with a combination of steady-state
The structure of tryptophan. 636 A
tons but small for the photons within a fluorescence photon burst. A series of photons is defined as a burst when the lag between a certain number of consecutive photons falls below a threshold value. The fluorescence lifetime is determined from a histogram of the fluorescence arrival times of the photons in a burst and can be used to identify molecules. Such identification is demonstrated with the fluorescent dyes Rhodamine B and Rhodamine 6G. The researchers demonstrate that quantification is also possible wiih single-molecule detection in an open volume element. Quantification is achieved by analyzing the burst size distribution of a sample survey. N.1V (the number of molecules in the sample) as determined by BIFL is in good agreement with the number determined by fluorescence correlation spectroscopy; both of those values differ from the expected value by a factor of only 2-3. The researchers explain this difference as the result of minor dilution errors (the dilution factor was 1:10") adsorption effects and
and time-resolved fluorescence techniques, photochemical isotope-exchange experiments, and transient absorption techniques. Some of the mechanisms that contribute to the nonradiative deactivation are intersystem crossing, solvent quenching, and excitedstate proton and electron transfer. Eight amino acid side chains—representing six functional groups—quench 3-methylindole fluorescence. The lysine e-amino group and the tyrosine phenol side chain quench by excited-state proton transfer. The side chains of glutamine, asparagine, glutamic and aspartic acid, cysteine, and histidine quench by excited-state electron transfer. The side chains of the remaining amino acids do not detectably quench 3-methylindole fluorescence. Bimolecular quenching studies indicate that lysine, glutamine, asparagine, and neutral histidine should be weak quenchers; neutral glutamic acid and aspartic acid should be moderate quenchers; and tyrosine, cysteine, positively charged histidine, and cystine should be strong quenchers. In an actual protein, other factors such as proximity, geometry, and local polarity will also affect the quenching process. Work is currently under way to extend their approach to intramolecular quenching in peptides. (Biochemistry 1 9 9 8 37 9976-82)
Analytical Chemistry News & Features, October 1, 1998
impurities in the solvent mixture. They also suggest that BIFL should be able to detect extremely small numbers of fluorescent molecules in the presence of an excess of dye molecules with other fluorescence properties. (J. Phys. Chem. A 1998,102, 6601-13)
Principle of BIFL spectroscopy with a two-dimensional time measurement and technique for burst selection and fluorescence lifetime determination.
Meaningful editing Solid-state NMR may be a technique with low sensitivity and broad line widths, but spectrometrists continue to invent clever ways to collect useful spectral data. In this case, Lyndon Emsley and co-workers at Ecole Normale Superieure de Lyon (France) and Bruker Analytik (Germany) introduce a solid-state NMR spectral editing technique that allows identification of heteronuclear peaks in powder samples containing compounds with natural abun-
One-dimensional CP spectrum (top) and SS-APT spectrum (bottom) of L-histidine monohydrochloride monohydrate.
