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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, Jr., 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

Original Manuscript: November 3, 2000
Revised Manuscript: February 28, 2001
Published: June 20, 2001

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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  1. R. G. Pinnick, S. C. Hill, P. Nachman, J. D. Pendleton, G. I. Fernandez, M. W. Mayo, J. G. Bruno, “Fluorescence particle counter for detecting airborne bacteria and other biological particles,” Aerosol Sci. Technol. 23, 653–664 (1995). [CrossRef]
  2. P. Nachman, G. Chen, R. G. Pinnick, S. C. Hill, R. K. Chang, M. W. Mayo, G. Fernandez, “Conditional-sampling spectrograph detection system for fluorescence measurements of individual airborne biological particles,” Appl. Opt. 35, 1069–1076 (1996). [CrossRef] [PubMed]
  3. P. P. Hairston, J. Ho, F. R. Quant, “Design of an instrument for real-time detection of bioaerosols using simultaneous measurement of particle aerodynamic size and intrinsic fluorescence,” J. Aerosol Sci. 28, 471–482 (1997). [CrossRef] [PubMed]
  4. R. G. Pinnick, S. C. Hill, P. Nachman, G. Videen, G. Chen, R. K. Chang, “Aerosol fluorescence spectrum analyzer for rapid measurement of single micrometer-sized airborne biological particles,” Aerosol Sci. Technol. 28, 95–104 (1998). [CrossRef]
  5. N. F. Fell, R. G. Pinnick, S. C. Hill, G. Videen, S. Niles, R. K. Chang, S. Holler, Y. Pan, J. Bottiger, B. V. Bronk, “Concentration, size, and excitation power effects on fluorescence from microdroplets and microparticles containing tryptophan and bacteria,” in Air Monitoring and Detection of Chemical and Biological Agents, J. Leonelli, M. L. Althouse, eds., Proc. SPIE3533, 52–63 (1998). [CrossRef]
  6. Y. S. Cheng, E. B. Barr, B. J. Fan, P. J. Hargis, D. J. Rader, T. J. O’Hern, J. R. Torczynski, G. C. Tisone, B. L. Preppernau, S. A. Young, R. J. Radloff, “Detection of bioaerosols using multiwavelength UV fluorescence spectroscopy,” Aerosol Sci. Technol. 30, 186–201 (1999). [CrossRef]
  7. Y. L. Pan, S. Holler, R. K. Chang, S. C. Hill, R. G. Pinnick, S. Niles, J. R. Bottiger, “Single-shot fluorescence spectra of individual micrometer-sized bioaerosols illuminated by a 351- or 266-nm ultraviolet laser,” Opt. Lett. 24, 116–119 (1999). [CrossRef]
  8. M. Seaver, J. D. Eversole, J. J. Hardgrove, W. K. Cary, D. C. Roselle, “Size and fluorescence measurements for field detection of biological aerosols,” Aerosol Sci. Technol. 30, 174–185 (1999). [CrossRef]
  9. F. L. Reyes, T. H. Jeys, N. R. Newbury, C. A. Primmerman, G. S. Rowe, A. Sanchez, “Bio-aerosol fluorescence sensor,” Field Anal. Chem. Technol. 3, 240–248 (1999). [CrossRef]
  10. J. D. Eversole, J. J. Hardgrove, W. K. Cary, D. P. Choulas, M. Seaver, “Continuous rapid biological aerosol detection with the use of UV fluorescence: outdoor test results,” Field Anal. Chem. Technol. 3, 249–259 (1999). [CrossRef]
  11. S. C. Hill, R. G. Pinnick, S. Niles, Y. Pan, S. Holler, R. K. Chang, J. R. Bottiger, B. T. Chen, C.-S. Orr, G. Feather, “Real-time measurement of fluorescence spectra from single airborne biological particles,” Field Anal. Chem. Technol. 3, 221–239 (1999). [CrossRef]
  12. P. H. Kaye, J. E. Barton, E. Hirst, J. M. Clark, “Simultaneous light scattering and intrinsic fluorescence measurement for the classification of airborne particles,” Appl. Opt. 39, 3738–3745 (2000). [CrossRef]
  13. M. F. Buehler, T. M. Allen, E. J. Davis, “Microparticle Raman spectroscopy of multicomponent aerosols,” J. Colloid Interface Sci. 146, 79–89 (1991). [CrossRef]
  14. G. Schweiger, “Raman scattering on single aerosol particles and on flowing aerosols: a review,” J. Aerosol Sci. 21, 483–509 (1990). [CrossRef]
  15. D. N. Whiteman, S. H. Melfi, “Cloud liquid water, mean droplet radius, and number density measurements using a Raman lidar,” J. Geophys. Res. 104, 31411–31419 (1999). [CrossRef]
  16. G. W. Faris, R. A. Copeland, K. Mortelmans, B. V. Bronk, “Spectrally resolved absolute fluorescence cross sections for bacillus spores,” Appl. Opt. 36, 958–967 (1997). [CrossRef] [PubMed]
  17. J. J. Tanke, P. van Oostvelt, P. van Duijn, “A parameter for the distribution of fluorophores in cells derived from measurements of inner filter effect and reabsorption phenomenon,” Cytometry 2, 359–369 (1982). [CrossRef] [PubMed]
  18. M. Kerker, M. A. Van Dilla, A. Brunsting, J. P. Kratohvil, P. Hsu, D. S. Wang, J. W. Gray, R. G. Langlois, “Is the central dogma of flow cytometry true: that fluorescence intensity is proportional to cellular dye content?” Cytometry 3, 71–78 (1982). [CrossRef] [PubMed]
  19. S. Hamada, S. Fujita, “Problem of size dependence in fluorescence DNA cytometry,” Cytometry 10, 394–401 (1989). [CrossRef] [PubMed]
  20. J. Eversole, H.-B. Lin, A. L. Huston, A. J. Campillo, P. T. Leung, S. Y. Lin, K. Young, “High-precision identification of morphology-dependent resonances in optical processes in microdroplets,” J. Opt. Soc. Am. B 10, 1955–1968 (1993). [CrossRef]
  21. J. Popp, M. Lankers, M. Trunk, I. Hartmann, E. Urlaub, W. Kiefer, “High-precision determination of size, refractive index, and dispersion of single microparticles from morphology-dependent resonances in optical processes,” Appl. Spectrosc. 52, 284–291 (1998). [CrossRef]
  22. J. Musick, J. Popp, M. Trunck, W. Kiefer, “Investigations of radical polymerization and copolymerization reactions in optically levitated microdroplets by simultaneous Raman spectroscopy, Mie scattering, and radiation pressure measurements,” Appl. Spectrosc. 52, 692–701 (1998). [CrossRef]
  23. H. Chew, P. J. McNulty, M. Kerker, “Model for Raman and fluorescent scattering by molecules embedded in small particles,” Phys. Rev. A 13, 396–404 (1976). [CrossRef]
  24. S. Druger, P. J. McNulty, “Radiation patterns of fluorescence from molecules embedded in small particles: general case,” Appl. Opt. 22, 75–82 (1983). [CrossRef] [PubMed]
  25. D. S. Wang, M. Kerker, H. Chew, “Raman and fluorescent scattering by molecules embedded in dielectric spheroids,” Appl. Opt. 19, 2315–2328 (1980). [CrossRef] [PubMed]
  26. S. C. Hill, V. Boutou, J. Yu, S. Ramstein, J.-P. Wolf, Y.-L. Pan, S. Holler, R. K. Chang, “Enhanced backward-directed multi-photon-excited fluorescence from dielectric microcavities,” Phys. Rev. Lett. 85, 54–57 (2000). [CrossRef] [PubMed]
  27. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983), p. 44.
  28. J. P. Kratohvil, M.-P. Lee, M. Kerker, “Angular distribution of fluorescence from small particles,” Appl. Opt. 17, 1978–1980 (1978). [CrossRef] [PubMed]
  29. E.-H. Lee, R. E. Benner, J. B. Fenn, R. K. Chang, “Angular distribution of fluorescence from monodispersed particles,” Appl. Opt. 17, 1980–1982 (1978). [CrossRef]
  30. H.-M. Tzeng, K. F. Wall, M. B. Long, R. K. Chang, “Laser emission from individual droplets at wavelengths corresponding to morphology-dependent resonances,” Opt. Lett. 9, 499–501 (1984). [CrossRef] [PubMed]
  31. H.-B. Lin, J. D. Eversole, A. J. Campillo, “Continuous-wave stimulated Raman scattering in microdroplets,” Opt. Lett. 17, 828–830 (1992). [CrossRef] [PubMed]
  32. H. Chew, “Radiation and lifetimes of atoms inside dielectric particles,” Phys. Rev. A 38, 3410–3416 (1988). [CrossRef] [PubMed]
  33. M. D. Barnes, W. B. Whitten, S. Arnold, J. M. Ramsey, “Homogeneous linewidths of Rhodamine 6G at room temperature from cavity-enhanced spontaneous emission rates,” J. Chem. Phys. 97, 7842–7845 (1992). [CrossRef]
  34. H.-B. Lin, J. D. Eversole, C. D. Merritt, A. J. Campillo, “Cavity-modified spontaneous-emission rates in liquid microdroplets,” Phys. Rev. A 45, 6756–6760 (1992). [CrossRef] [PubMed]
  35. M. D. Barnes, C.-Y. Kung, W. B. Whitten, J. M. Ramsey, S. Arnold, S. Holler, “Fluorescence of oriented molecules in a microcavity,” Phys. Rev. Lett. 76, 3931–3934 (1996). [CrossRef] [PubMed]
  36. M. D. Barnes, W. B. Whitten, J. M. Ramsey, “Enhanced fluorescence yields through cavity-QED effects in microdroplets,” J. Opt. Soc. Am. B 11, 1297–1304 (1994). [CrossRef]
  37. D. Creed, “The photophysics and photochemistry of the near-UV absorbing amino acids. I. Tryptophan and its simple derivatives,” Photochem. Photobiol. 39, 537–562 (1984). [CrossRef]
  38. Y.-L. Pan, R. G. Pinnick, S. C. Hill, S. Niles, S. Holler, J. R. Bottiger, J.-P. Wolf, R. K. Chang, “Dynamics of photon-induced degradation and fluorescence in riboflavin microparticles,” Appl. Phys. B 72, 449–454 (2001). [CrossRef]
  39. D. Q. Chowdhury, S. C. Hill, M. Mazumder, “Absorptive bistability in a dielectric sphere,” Opt. Commun. 131, 343–346 (1996). [CrossRef]
  40. P. W. Barber, S. C. Hill, Light Scattering by Particles: Computational Methods (World Scientific, Singapore, 1990), Chap. 4.
  41. M. I. Mishchenko, J. W. Hovenier, L. D. Travis, Light Scattering by Nonspherical Particles: Theory, Measurements, and Applications (Academic, San Diego, Calif., 2000).
  42. J. R. Bottiger, P. J. Deluca, E. W. Stuebing, D. R. VanReenen, “An ink jet aerosol generator,” J. Aerosol Sci. 29(Suppl. 1), S965–S966 (1998). [CrossRef]
  43. H.-M. Tzeng, K. F. Wall, M. B. Long, R. K. Chang, “Evaporation and condensation rates of liquid droplets deduced from structure resonances in the fluorescence spectra,” Opt. Lett. 9, 273–275 (1984). [CrossRef] [PubMed]
  44. A quantum efficiency of 0.15 is in the range of reported values for tryptophan in water45 and for class B proteins (proteins that contain tryptophan).46 See also Ref. 16, p. 964, and references cited therein.
  45. I. Weinryb, R. F. Steiner, “The luminescence of the aromatic amino acids,” in Excited States of Proteins and Nucleic Acids, R. F. Steiner, I. Weinryb, eds. (Plenum, New York, 1971), Table 2, pp. 289–290.
  46. J. W. Longworth, “Luminescence of polypeptides and proteins,” in Excited States of Proteins and Nucleic Acids, R. F. Steiner, I. Weinryb, eds. (Plenum, New York, 1971), Table 10, p. 434.
  47. C. F. Bohren, D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley InterScience, New York, 1983), Chap. 4.
  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.
  52. W. G. Murrell, “Chemical composition of spores and spore structures,” in The Bacterial Spore, A. Hurst, G. W. Gould, eds. (Academic, New York, 1969), pp. 218–231; Table III, p. 221.
  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.
  55. S. J. Isak, E. M. Eyring, “Fluorescence quantum yield of cresyl violet in methanol and water as a function of concentration,” J. Phys. Chem. 96, 1738–1742 (1992). [CrossRef]

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