Reproducible fluorescence spectra of individual 2- to 5-microm -diameter biological aerosol particles excited with a single shot from a Q -switched laser (266 or 351 nm) have been obtained with highly improved signal-to-noise ratios. Critical to the advance are crossed diode-laser trigger beams, which precisely define the sample volume, and a reflecting objective, which minimizes chromatic aberration and has a large N.A. for collecting fluorescence. Several allergens (red oak, meadow oat pollen, paper mulberry pollen, and puffball spores) have different fluorescence spectra. Bacillus subtilis fluorescence spectrum deteriorates at high 266-nm incident intensity. Dry riboflavin particles illuminated with a 351-nm light exhibit a new 420-nm fluorescence peak that grows nonlinearly with laser pulse energy.
We measured fluorescence from spherical water droplets containing tryptophan and from aggregates of bacterial cells and compared these measurements with calculations of fluorescence of dielectric spheres. The measured dependence of fluorescence on size, from both droplets and dry-particle aggregates of bacteria, is proportional to the absorption cross section calculated for homogeneous spheres containing the appropriate percentage of tryptophan. However, as the tryptophan concentration of the water droplets is increased, the measured fluorescence from droplets increases less than predicted, probably because of concentration quenching. We model the dependence of the fluorescence on input intensity by assuming that the average time between fluorescence emission events is the sum of the fluorescence lifetime and the excitation lifetime (the average time it takes for an illuminated molecule to be excited), which we calculated assuming that the intensity inside the particle is uniform. Even though the intensity inside the particles spatially varies, this assumption of uniform intensity still leads to results consistent with the measured intensity dependence.
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