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Applied Optics

Applied Optics


  • Vol. 40, Iss. 18 — Jun. 20, 2001
  • pp: 3005–3013

Fluorescence from Airborne Microparticles: Dependence on Size, Concentration of Fluorophores, and Illumination Intensity

Steven C. Hill, Ronald G. Pinnick, Stanley Niles, Nicholas F. Fell, Yong-Le Pan, Jerold Bottiger, Burt V. Bronk, Stephen Holler, and Richard K. Chang  »View Author Affiliations

Applied Optics, Vol. 40, Issue 18, pp. 3005-3013 (2001)

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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.

© 2001 Optical Society of America

OCIS Codes
(010.1100) Atmospheric and oceanic optics : Aerosol detection
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(290.4020) Scattering : Mie theory
(290.5850) Scattering : Scattering, particles
(290.5860) Scattering : Scattering, Raman
(300.2530) Spectroscopy : Fluorescence, laser-induced

Steven C. Hill, Ronald G. Pinnick, Stanley Niles, Nicholas F. Fell, Yong-Le Pan, Jerold Bottiger, Burt V. Bronk, Stephen Holler, and Richard K. Chang, "Fluorescence from Airborne Microparticles: Dependence on Size, Concentration of Fluorophores, and Illumination Intensity," Appl. Opt. 40, 3005-3013 (2001)

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  48. This value is obtained by calculation from the absorptivity given in Fig. 11.1 of Ref. 27, p. 343, and is essentially the same as the 2.1 × 10−17 cm2 at 274 nm used in Ref. 16, p. 964 (see references cited therein).
  49. A lifetime of 3 ns is in the range of reported values for tryptophan in water45 and for class B proteins.46
  50. Although the shot-to-shot variability of the spectra can be quite good (see, e.g., Fig. 2 of Ref. 7 and Fig. 4 of Ref. 11 for examples with 5-μm- and 4-μm-diameter particles, respectively), the accumulated spectra allow a more thorough comparison because they have smaller shot-to-shot variations. Some causes of these variations are spatial and shot-to-shot variations in the laser beam, variations in particle trajectories and in particle sizes, and detector noise.
  51. The calculated results shown in Fig. 3(a) are remarkably insensitive to variations in the real part of the refractive index over a large range (e.g., 1.3 < mr < 1.7). Therefore, even though we do not know the average refractive index of these inhomogeneous particles, the calculated results would be essentially the same when any mr is used in the range of possible values. The percentage dry weight of tryptophan is more problematical. The fluorescence properties of tryptophan depend on their local environment, which can be different for different tryptophan molecules even in the same protein.46 Dry weights of tryptophan in B. subitis have been reported as 3% in vegetative cells and 5% in spores.52 The reason we assume 4% tryptophan for these vegetative cells is that such cells are also reported to contain 3.5% tyrosine, which also absorbs 266-nm light (but less efficiently than tryptophan), and which can transfer the absorbed energy to tryptophan.46 A more accurate model might include a calculation of the contribution to the imaginary part of the refractive index [as in Eq. (1)] from each species of fluorophore in the particle. This would also account more rigorously for energy transfer between molecules; however, that is beyond the scope of this paper and probably beyond the accuracy of our measurements. The shape of the calculated curve in Fig. 3(a) is somewhat sensitive to the concentration of tryptophan. However, our data do not appear accurate enough to distinguish between 2% and 4% tryptophan. A further limitation is that the B. subtilis was used as purchased, and we do not know the purity of the sample.
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  53. The calculations in Ref. 26 are for droplets in which the dye molecule rotation times are short compared to fluorescence lifetimes. However, we do not expect the rotation time to cause a major shift in the size at which the angular fluorescence becomes size independent.
  54. P. Pringsheim, Fluorescence and Phosphorescence (Interscience, New York, 1949), pp. 347–353.
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