Abstract

We report the design and operation of a prototype conditional-sampling spectrograph detection system that can record the fluorescence spectra of individual, micrometer-sized aerosols as they traverse an intense 488-nm intracavity laser beam. The instrument's image-intensified CCD detector is gated by elastic scattering or by undispersed fluorescence from particles that enter the spectrograph's field of view. It records spectra only from particles with preselected scattering–fluorescence levels (a fiber-optic–photomultiplier subsystem provides the gating signal). This conditional-sampling procedure reduces data-handling rates and increases the signal-to-noise ratio by restricting the system's exposures to brief periods when aerosols traverse the beam. We demonstrate these advantages by reliably capturing spectra from individual fluorescent microspheres dispersed in an airstream. The conditional-sampling procedure also permits some discrimination among different types of particles, so that spectra may be recorded from the few interesting particles present in a cloud of background aerosol. We demonstrate such discrimination by measuring spectra from selected fluorescent microspheres in a mixture of two types of microspheres, and from bacterial spores in a mixture of spores and nonfluorescent kaolin particles.

© 1996 Optical Society of America

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  1. J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).
  2. J. M. Prospero, D. L. Savoie, R. T. Nees, R. A. Duce, J. Merrill, “Particulate sulfate and nitrate in the boundary layer over the north Pacific Ocean,” J. Geophys. Res. 90, 10,586–10,596 (1985).
  3. R. E. Weiss, A. P. Waggoner, R. J. Charlson, N. C. Ahlquist, “Sulfate aerosol: its geographical extent in the midwestern and southern United States,” Science 195, 979–981 (1977).
  4. S. Twomey, Atmospheric Aerosols (Elsevier, New York, 1977), Chap. 2, pp. 23–45.
  5. B. Lighthart, L. D. Stetzenbach, “Distribution of microbial bioaerosol,” in Atmospheric Microbial Aerosols, B. Lighthart, G. Mohr, eds. (Chapman & Hall, New York, 1994), Chap. 4, pp. 68–98.
  6. T. C. Eikhoff, “Perspectives on airborne infections in health care facilities,” in Proceedings of the Workshop on Engineering Controls for Preventing Airborne Infections in Workers in Health Care and Related Facilities, P. J. Bierbaum, M. Lippmann, eds. (National Institute of Occupational Safety and Health, Cincinnati, 1994), Pub. 94–106, pp. 15–34;Stockholm International Peace Research Institute, The Problem of Chemical and Biological Weapons, Vol. 1: The Rise of CB Weapons (Almqvist & Wiksell, Stockholm, 1971), pp. 111–124;U.S. Congress, Office of Technology Assessment, Technologies Underlying Weapons of Mass Destruction (U.S. GPO, Washington, D.C., 1993), Pub. OTA-BP-ISC-115, pp. 71–117.
  7. J. E. Aubin, “Autofluorescence of viable cultured mammalian cells,” J. Histochem. Cytochem. 27, 26–43 (1979).
  8. R. C. Benson, R. A. Meyer, M. E. Zaruba, G. M. McKhann, “Cellular autofluorescence: is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).
  9. M. J. Sorrell, J. Tribble, L. Reinisch, “Bacteria identification of otitis media with fluorescence spectroscopy,” Lasers Surg. Med. 14, 155–163 (1994);J. A. Werkhaven, L. Reinisch, M. J. Sorrell, J. Tribble, R. H. Ossoff, “Noninvasive optical diagnosis of bacteria causing otitis media,” Laryngoscope 104, 264–268 (1994);R. A. Dalterio, W. H. Nelson, D. Britt, J. F. Sperry, D. Psaras, J. F. Tanguay, S. L. Suib, “Steady-state and decay characteristics of protein tryptophan fluorescence from bacteria,” Appl. Spectrosc. 40, 86–90 (1986);B. V. Bronk, L. Reinisch, “Variability of steady-state bacterial fluorescence with respect to growth conditions,” Appl. Spectrosc. 47, 436–440 (1993);J. Ho, G. Fisher, “Detection of BW agents: flow cytometry measurement of Bacillus subtilis (BG) spore fluorescence,” Memo. 1421 (Defense Research Establishment, Suffield, Medicine Hat, Alberta, Canada, 1993).
  10. J. Gelbwachs, M. Birnbaum, “Fluorescence of atmospheric aerosols and lidar implications,” Appl. Opt. 12, 2442–2447 (1973).
  11. R. G. Pinnick, S. C. Hill, P. Nachman, J. D. Pendleton, G. L. Fernandez, M. W. Mayo, J. G. Bruno, “Fluorescence particle counter for detecting airborne bacteria and other biological particles,” Aerosol Sci. Technol. (to be published).
  12. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983), pp. 14–15.
  13. S. C. Hill, R. G. Pinnick, P. Nachman, G. Chen, R. K. Chang, M. W. Mayo, G. L. Fernandez, “Aerosol-fluorescence spectrum analyzer: real-time measurement of emission spectra of individual airborne bacteria, pollens, and other particles,” Appl. Opt. 34, 7149–7155 (1995).This instrument was able, over a limited range of flow conditions, to obtain spectra from individual dye-doped particles in an airstream.
  14. Our conditional-sampling technique differs from familiar gating methods, such as boxcar integration. Conditional sampling operates on demand at the random times that interesting signals are present; in the usual gating methods, the detection systems are triggered in coincidence with phenomena that are also triggered.
  15. P. Setlow, “Germination and outgrowth,” in The Bacterial Spore, A. Hurst, G. W. Gould, eds. (Academic, London, 1983), p. 214.
  16. J. B. Perkins, J. G. Pero, “Biosynthesis of riboflavin, biotin, folic acid and cobalamin,” in Bacillus Subtilis and Other Gram Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, A. L. Sonensheim, J. A. Hoch, R. Losick, eds. (American Society for Microbiology, Washington, D.C., 1993), pp. 319–334.
  17. We positioned the intracavity lens and the closure mirror so that, within the laser's gain medium, the extended cavity's circulating mode is the same as the original cavity's mode. The 25-cm focal length, plano–convex lens is made of fused silica. (We found that a comparable lens made of BK-7 glass, used in our initial study with the extended cavity, showed thermally induced distortion, caused by the high-power circulating intracavity. The fused-silica lens has much smaller thermal effects.) Wavelength selection is accomplished by the laser's intracavity prism and mode-defining aperture, still in their original locations. However, the wavelength discrimination is less effective in the modified configuration, so ~20% of the extended cavity's circulating power is actually in two additional laser lines (at 476.5 and 496.5 nm) adjacent to the dominant 488-nm line.
  18. We did not use the PMS scattering cell-flow system intact because the light gathered by its specialized collection optics (a paraboloidal mirror segment covering more than 2π sr around the intersection volume of laser beam and aerosol stream) could not be focused to an image of sufficient quality for our purpose.
  19. We were forced to collect light at 30° by our improvised flow system. This geometry may be suboptimal for detecting weak fluorescence in the presence of strong elastic scattering. However, because our setup was intracavity, we were also collecting light at 150° to the backward excitation beam; this may be a relatively favorable geometry for avoiding elastically scattered light. In any event, our spectra appear to be uncontaminated by elastic light.
  20. The fiber-optics-coupled image-intensified CCD detector was from Princeton Instruments (Model ICCD-576ES). Its image intensifier was gated by a high-voltage pulser (Model FG-100). A controller (Model ST-130) read out and digitized signals from the CCD detector and sent them to our computer. The detector incorporates a CCD chip made by EEV, Ltd. (Chelmsford, Essex, U.K.) that has 22-μm-square pixels in an array that is 576 elements (horizontal) by 384 elements (vertical).
  21. The fused-silica fiber has cladding, buffer, and jacket diameters of 660, 690, and 1200 μm, respectively. Its numerical aperture is 0.22. The fiber assembly was purchased from Polymicro Technologies, Inc., Phoenix, Ariz. 85023.
  22. We also placed a long-pass (~500-nm) colored-glass filter just outside the spectrograph's entrance slit to attenuate the very bright elastically scattered light at 488 nm, thus minimizing stray light levels within the spectrograph and protecting the detector array's intensifier from possible damage. However, this filter transmitted enough at 488 nm to yield usable pulses in the elastic triggering channel.
  23. AStanford Research DG535 digital delay generator is used in each channel to establish the voltage threshold for input triggering pulses and to set the time delay on the resulting transistor–transistor logic output pulses.
  24. All spectra reported here were taken with an entrance slit width of 200 μm. The spectra were binned vertically (i.e., all the pixels in a vertical column of the CCD were summed to integrate all the captured light at each wavelength). The microchannel-plate intensifier was used with accelerating potentials between 800 and 900 V.
  25. Duke Scientific Corp., 2463 Faber Place, Palo Alto, Calif. 94303.
  26. R. E. Benner, P. W. Barber, J. F. Owen, R. K. Chang, “Observation of structure resonances in the fluorescence spectra from microspheres,” Phys. Rev. Lett. 44, 475–479 (1980);A. J. Campillo, J. D. Eversole, H. B. Lin, “Cavity quantum electrodynamic enhancement of stimulated emission in microdroplets,” Phys. Rev. Lett. 67, 437–441 (1991);M. D. Barnes, W. B. Whitten, J. M. Ramsey, “Enhanced fluorescence yields through cavity-quantum electrodynamic effects in microspheres,” J. Chem. Phys. 97, 7842–7845 (1992).
  27. P. Chylek, “Partial-wave resonances and the ripple structure in the Mie normalized extinction cross section,” J. Opt. Soc. Am. 66, 285–287 (1976).
  28. P. Conwell, C. K. Rushforth, R. E. Benner, S. C. Hill, “Efficient automated algorithm for the sizing of dielectric microspheres using the resonance spectrum,”; J. Opt. Soc. Am. A 1, 1680–1687 (1984).
  29. J. D. Eversole, H. B. Lin, A. L. Huston, A. J. Campillo, P. T. Leung, S. Y. Liu, K. Young, “High precision identification of morphology dependent resonances in optical processes in microdroplets,”; J. Opt. Soc. Am. B 10, 1955–1968 (1993).
  30. The wavelength spacings of spectral peaks from the green-yellow fluorescing microspheres (4.5-μm diameter) are closer than the spacings from the pink-fluorescing spheres (1.96-μm diameter), because spacings decrease with particle size; see Ref. 27. Thus use of the two sphere types provides two independent confirmations of the conditional-sampling system's discrimination abilities, i.e., by means of peak spacings and by means of spectral region of fluoresence.
  31. Likely noise sources in the PMT's are shot noise on their dark currents and on any dc-background light level. (The dc-background current would not have been evident to us, because the transimpedance amplifiers have ac-coupled outputs.)
  32. R. K. Reich, R. W. Mountain, W. H. McGonagle, J. C. M. Huang, J. C. Twichell, B. B. Kosicki, E. D. Savoye, “Integrated electronic shutter for back-illuminated chargecoupled devices,” IEEE Trans. Electron Devices 40, 1231–1235 (1993).
  33. M. J. Padgett, A. R. Harvey, A. J. Duncan, W. Sibbett, “Single-pulse, Fourier-transform spectrometer having no moving parts,” Appl. Opt. 33, 6035–6039 (1994).

1995

1994

M. J. Sorrell, J. Tribble, L. Reinisch, “Bacteria identification of otitis media with fluorescence spectroscopy,” Lasers Surg. Med. 14, 155–163 (1994);J. A. Werkhaven, L. Reinisch, M. J. Sorrell, J. Tribble, R. H. Ossoff, “Noninvasive optical diagnosis of bacteria causing otitis media,” Laryngoscope 104, 264–268 (1994);R. A. Dalterio, W. H. Nelson, D. Britt, J. F. Sperry, D. Psaras, J. F. Tanguay, S. L. Suib, “Steady-state and decay characteristics of protein tryptophan fluorescence from bacteria,” Appl. Spectrosc. 40, 86–90 (1986);B. V. Bronk, L. Reinisch, “Variability of steady-state bacterial fluorescence with respect to growth conditions,” Appl. Spectrosc. 47, 436–440 (1993);J. Ho, G. Fisher, “Detection of BW agents: flow cytometry measurement of Bacillus subtilis (BG) spore fluorescence,” Memo. 1421 (Defense Research Establishment, Suffield, Medicine Hat, Alberta, Canada, 1993).

M. J. Padgett, A. R. Harvey, A. J. Duncan, W. Sibbett, “Single-pulse, Fourier-transform spectrometer having no moving parts,” Appl. Opt. 33, 6035–6039 (1994).

1993

J. D. Eversole, H. B. Lin, A. L. Huston, A. J. Campillo, P. T. Leung, S. Y. Liu, K. Young, “High precision identification of morphology dependent resonances in optical processes in microdroplets,”; J. Opt. Soc. Am. B 10, 1955–1968 (1993).

R. K. Reich, R. W. Mountain, W. H. McGonagle, J. C. M. Huang, J. C. Twichell, B. B. Kosicki, E. D. Savoye, “Integrated electronic shutter for back-illuminated chargecoupled devices,” IEEE Trans. Electron Devices 40, 1231–1235 (1993).

1985

J. M. Prospero, D. L. Savoie, R. T. Nees, R. A. Duce, J. Merrill, “Particulate sulfate and nitrate in the boundary layer over the north Pacific Ocean,” J. Geophys. Res. 90, 10,586–10,596 (1985).

1984

P. Conwell, C. K. Rushforth, R. E. Benner, S. C. Hill, “Efficient automated algorithm for the sizing of dielectric microspheres using the resonance spectrum,”; J. Opt. Soc. Am. A 1, 1680–1687 (1984).

1983

J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).

1980

R. E. Benner, P. W. Barber, J. F. Owen, R. K. Chang, “Observation of structure resonances in the fluorescence spectra from microspheres,” Phys. Rev. Lett. 44, 475–479 (1980);A. J. Campillo, J. D. Eversole, H. B. Lin, “Cavity quantum electrodynamic enhancement of stimulated emission in microdroplets,” Phys. Rev. Lett. 67, 437–441 (1991);M. D. Barnes, W. B. Whitten, J. M. Ramsey, “Enhanced fluorescence yields through cavity-quantum electrodynamic effects in microspheres,” J. Chem. Phys. 97, 7842–7845 (1992).

1979

J. E. Aubin, “Autofluorescence of viable cultured mammalian cells,” J. Histochem. Cytochem. 27, 26–43 (1979).

R. C. Benson, R. A. Meyer, M. E. Zaruba, G. M. McKhann, “Cellular autofluorescence: is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).

1977

R. E. Weiss, A. P. Waggoner, R. J. Charlson, N. C. Ahlquist, “Sulfate aerosol: its geographical extent in the midwestern and southern United States,” Science 195, 979–981 (1977).

1976

P. Chylek, “Partial-wave resonances and the ripple structure in the Mie normalized extinction cross section,” J. Opt. Soc. Am. 66, 285–287 (1976).

1973

Ahlquist, N. C.

R. E. Weiss, A. P. Waggoner, R. J. Charlson, N. C. Ahlquist, “Sulfate aerosol: its geographical extent in the midwestern and southern United States,” Science 195, 979–981 (1977).

Aubin, J. E.

J. E. Aubin, “Autofluorescence of viable cultured mammalian cells,” J. Histochem. Cytochem. 27, 26–43 (1979).

Barber, P. W.

R. E. Benner, P. W. Barber, J. F. Owen, R. K. Chang, “Observation of structure resonances in the fluorescence spectra from microspheres,” Phys. Rev. Lett. 44, 475–479 (1980);A. J. Campillo, J. D. Eversole, H. B. Lin, “Cavity quantum electrodynamic enhancement of stimulated emission in microdroplets,” Phys. Rev. Lett. 67, 437–441 (1991);M. D. Barnes, W. B. Whitten, J. M. Ramsey, “Enhanced fluorescence yields through cavity-quantum electrodynamic effects in microspheres,” J. Chem. Phys. 97, 7842–7845 (1992).

Benner, R. E.

P. Conwell, C. K. Rushforth, R. E. Benner, S. C. Hill, “Efficient automated algorithm for the sizing of dielectric microspheres using the resonance spectrum,”; J. Opt. Soc. Am. A 1, 1680–1687 (1984).

R. E. Benner, P. W. Barber, J. F. Owen, R. K. Chang, “Observation of structure resonances in the fluorescence spectra from microspheres,” Phys. Rev. Lett. 44, 475–479 (1980);A. J. Campillo, J. D. Eversole, H. B. Lin, “Cavity quantum electrodynamic enhancement of stimulated emission in microdroplets,” Phys. Rev. Lett. 67, 437–441 (1991);M. D. Barnes, W. B. Whitten, J. M. Ramsey, “Enhanced fluorescence yields through cavity-quantum electrodynamic effects in microspheres,” J. Chem. Phys. 97, 7842–7845 (1992).

Benson, R. C.

R. C. Benson, R. A. Meyer, M. E. Zaruba, G. M. McKhann, “Cellular autofluorescence: is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).

Birnbaum, M.

Bruno, J. G.

R. G. Pinnick, S. C. Hill, P. Nachman, J. D. Pendleton, G. L. Fernandez, M. W. Mayo, J. G. Bruno, “Fluorescence particle counter for detecting airborne bacteria and other biological particles,” Aerosol Sci. Technol. (to be published).

Campillo, A. J.

Chang, R. K.

S. C. Hill, R. G. Pinnick, P. Nachman, G. Chen, R. K. Chang, M. W. Mayo, G. L. Fernandez, “Aerosol-fluorescence spectrum analyzer: real-time measurement of emission spectra of individual airborne bacteria, pollens, and other particles,” Appl. Opt. 34, 7149–7155 (1995).This instrument was able, over a limited range of flow conditions, to obtain spectra from individual dye-doped particles in an airstream.

R. E. Benner, P. W. Barber, J. F. Owen, R. K. Chang, “Observation of structure resonances in the fluorescence spectra from microspheres,” Phys. Rev. Lett. 44, 475–479 (1980);A. J. Campillo, J. D. Eversole, H. B. Lin, “Cavity quantum electrodynamic enhancement of stimulated emission in microdroplets,” Phys. Rev. Lett. 67, 437–441 (1991);M. D. Barnes, W. B. Whitten, J. M. Ramsey, “Enhanced fluorescence yields through cavity-quantum electrodynamic effects in microspheres,” J. Chem. Phys. 97, 7842–7845 (1992).

Charlson, R. J.

J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).

R. E. Weiss, A. P. Waggoner, R. J. Charlson, N. C. Ahlquist, “Sulfate aerosol: its geographical extent in the midwestern and southern United States,” Science 195, 979–981 (1977).

Chen, G.

Chylek, P.

P. Chylek, “Partial-wave resonances and the ripple structure in the Mie normalized extinction cross section,” J. Opt. Soc. Am. 66, 285–287 (1976).

Conwell, P.

P. Conwell, C. K. Rushforth, R. E. Benner, S. C. Hill, “Efficient automated algorithm for the sizing of dielectric microspheres using the resonance spectrum,”; J. Opt. Soc. Am. A 1, 1680–1687 (1984).

Delany, A. C.

J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).

Duce, R. A.

J. M. Prospero, D. L. Savoie, R. T. Nees, R. A. Duce, J. Merrill, “Particulate sulfate and nitrate in the boundary layer over the north Pacific Ocean,” J. Geophys. Res. 90, 10,586–10,596 (1985).

Duncan, A. J.

Eikhoff, T. C.

T. C. Eikhoff, “Perspectives on airborne infections in health care facilities,” in Proceedings of the Workshop on Engineering Controls for Preventing Airborne Infections in Workers in Health Care and Related Facilities, P. J. Bierbaum, M. Lippmann, eds. (National Institute of Occupational Safety and Health, Cincinnati, 1994), Pub. 94–106, pp. 15–34;Stockholm International Peace Research Institute, The Problem of Chemical and Biological Weapons, Vol. 1: The Rise of CB Weapons (Almqvist & Wiksell, Stockholm, 1971), pp. 111–124;U.S. Congress, Office of Technology Assessment, Technologies Underlying Weapons of Mass Destruction (U.S. GPO, Washington, D.C., 1993), Pub. OTA-BP-ISC-115, pp. 71–117.

Eversole, J. D.

Fernandez, G. L.

Gelbwachs, J.

Harvey, A. R.

Hill, S. C.

S. C. Hill, R. G. Pinnick, P. Nachman, G. Chen, R. K. Chang, M. W. Mayo, G. L. Fernandez, “Aerosol-fluorescence spectrum analyzer: real-time measurement of emission spectra of individual airborne bacteria, pollens, and other particles,” Appl. Opt. 34, 7149–7155 (1995).This instrument was able, over a limited range of flow conditions, to obtain spectra from individual dye-doped particles in an airstream.

P. Conwell, C. K. Rushforth, R. E. Benner, S. C. Hill, “Efficient automated algorithm for the sizing of dielectric microspheres using the resonance spectrum,”; J. Opt. Soc. Am. A 1, 1680–1687 (1984).

R. G. Pinnick, S. C. Hill, P. Nachman, J. D. Pendleton, G. L. Fernandez, M. W. Mayo, J. G. Bruno, “Fluorescence particle counter for detecting airborne bacteria and other biological particles,” Aerosol Sci. Technol. (to be published).

Hohnen, V.

J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).

Huang, J. C. M.

R. K. Reich, R. W. Mountain, W. H. McGonagle, J. C. M. Huang, J. C. Twichell, B. B. Kosicki, E. D. Savoye, “Integrated electronic shutter for back-illuminated chargecoupled devices,” IEEE Trans. Electron Devices 40, 1231–1235 (1993).

Huston, A. L.

Jaenicke, R.

J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).

Kosicki, B. B.

R. K. Reich, R. W. Mountain, W. H. McGonagle, J. C. M. Huang, J. C. Twichell, B. B. Kosicki, E. D. Savoye, “Integrated electronic shutter for back-illuminated chargecoupled devices,” IEEE Trans. Electron Devices 40, 1231–1235 (1993).

Lakowicz, J. R.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983), pp. 14–15.

Leung, P. T.

Lighthart, B.

B. Lighthart, L. D. Stetzenbach, “Distribution of microbial bioaerosol,” in Atmospheric Microbial Aerosols, B. Lighthart, G. Mohr, eds. (Chapman & Hall, New York, 1994), Chap. 4, pp. 68–98.

Lin, H. B.

Liu, S. Y.

Mayo, M. W.

McGonagle, W. H.

R. K. Reich, R. W. Mountain, W. H. McGonagle, J. C. M. Huang, J. C. Twichell, B. B. Kosicki, E. D. Savoye, “Integrated electronic shutter for back-illuminated chargecoupled devices,” IEEE Trans. Electron Devices 40, 1231–1235 (1993).

McKhann, G. M.

R. C. Benson, R. A. Meyer, M. E. Zaruba, G. M. McKhann, “Cellular autofluorescence: is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).

Merrill, J.

J. M. Prospero, D. L. Savoie, R. T. Nees, R. A. Duce, J. Merrill, “Particulate sulfate and nitrate in the boundary layer over the north Pacific Ocean,” J. Geophys. Res. 90, 10,586–10,596 (1985).

Meyer, R. A.

R. C. Benson, R. A. Meyer, M. E. Zaruba, G. M. McKhann, “Cellular autofluorescence: is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).

Mountain, R. W.

R. K. Reich, R. W. Mountain, W. H. McGonagle, J. C. M. Huang, J. C. Twichell, B. B. Kosicki, E. D. Savoye, “Integrated electronic shutter for back-illuminated chargecoupled devices,” IEEE Trans. Electron Devices 40, 1231–1235 (1993).

Moyers, J.

J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).

Nachman, P.

Nees, R. T.

J. M. Prospero, D. L. Savoie, R. T. Nees, R. A. Duce, J. Merrill, “Particulate sulfate and nitrate in the boundary layer over the north Pacific Ocean,” J. Geophys. Res. 90, 10,586–10,596 (1985).

Owen, J. F.

R. E. Benner, P. W. Barber, J. F. Owen, R. K. Chang, “Observation of structure resonances in the fluorescence spectra from microspheres,” Phys. Rev. Lett. 44, 475–479 (1980);A. J. Campillo, J. D. Eversole, H. B. Lin, “Cavity quantum electrodynamic enhancement of stimulated emission in microdroplets,” Phys. Rev. Lett. 67, 437–441 (1991);M. D. Barnes, W. B. Whitten, J. M. Ramsey, “Enhanced fluorescence yields through cavity-quantum electrodynamic effects in microspheres,” J. Chem. Phys. 97, 7842–7845 (1992).

Padgett, M. J.

Pendleton, J. D.

R. G. Pinnick, S. C. Hill, P. Nachman, J. D. Pendleton, G. L. Fernandez, M. W. Mayo, J. G. Bruno, “Fluorescence particle counter for detecting airborne bacteria and other biological particles,” Aerosol Sci. Technol. (to be published).

Perkins, J. B.

J. B. Perkins, J. G. Pero, “Biosynthesis of riboflavin, biotin, folic acid and cobalamin,” in Bacillus Subtilis and Other Gram Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, A. L. Sonensheim, J. A. Hoch, R. Losick, eds. (American Society for Microbiology, Washington, D.C., 1993), pp. 319–334.

Pero, J. G.

J. B. Perkins, J. G. Pero, “Biosynthesis of riboflavin, biotin, folic acid and cobalamin,” in Bacillus Subtilis and Other Gram Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, A. L. Sonensheim, J. A. Hoch, R. Losick, eds. (American Society for Microbiology, Washington, D.C., 1993), pp. 319–334.

Pinnick, R. G.

Prospero, J. M.

J. M. Prospero, D. L. Savoie, R. T. Nees, R. A. Duce, J. Merrill, “Particulate sulfate and nitrate in the boundary layer over the north Pacific Ocean,” J. Geophys. Res. 90, 10,586–10,596 (1985).

J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).

Rahn, K.

J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).

Reich, R. K.

R. K. Reich, R. W. Mountain, W. H. McGonagle, J. C. M. Huang, J. C. Twichell, B. B. Kosicki, E. D. Savoye, “Integrated electronic shutter for back-illuminated chargecoupled devices,” IEEE Trans. Electron Devices 40, 1231–1235 (1993).

Reinisch, L.

M. J. Sorrell, J. Tribble, L. Reinisch, “Bacteria identification of otitis media with fluorescence spectroscopy,” Lasers Surg. Med. 14, 155–163 (1994);J. A. Werkhaven, L. Reinisch, M. J. Sorrell, J. Tribble, R. H. Ossoff, “Noninvasive optical diagnosis of bacteria causing otitis media,” Laryngoscope 104, 264–268 (1994);R. A. Dalterio, W. H. Nelson, D. Britt, J. F. Sperry, D. Psaras, J. F. Tanguay, S. L. Suib, “Steady-state and decay characteristics of protein tryptophan fluorescence from bacteria,” Appl. Spectrosc. 40, 86–90 (1986);B. V. Bronk, L. Reinisch, “Variability of steady-state bacterial fluorescence with respect to growth conditions,” Appl. Spectrosc. 47, 436–440 (1993);J. Ho, G. Fisher, “Detection of BW agents: flow cytometry measurement of Bacillus subtilis (BG) spore fluorescence,” Memo. 1421 (Defense Research Establishment, Suffield, Medicine Hat, Alberta, Canada, 1993).

Rushforth, C. K.

P. Conwell, C. K. Rushforth, R. E. Benner, S. C. Hill, “Efficient automated algorithm for the sizing of dielectric microspheres using the resonance spectrum,”; J. Opt. Soc. Am. A 1, 1680–1687 (1984).

Savoie, D. L.

J. M. Prospero, D. L. Savoie, R. T. Nees, R. A. Duce, J. Merrill, “Particulate sulfate and nitrate in the boundary layer over the north Pacific Ocean,” J. Geophys. Res. 90, 10,586–10,596 (1985).

Savoye, E. D.

R. K. Reich, R. W. Mountain, W. H. McGonagle, J. C. M. Huang, J. C. Twichell, B. B. Kosicki, E. D. Savoye, “Integrated electronic shutter for back-illuminated chargecoupled devices,” IEEE Trans. Electron Devices 40, 1231–1235 (1993).

Setlow, P.

P. Setlow, “Germination and outgrowth,” in The Bacterial Spore, A. Hurst, G. W. Gould, eds. (Academic, London, 1983), p. 214.

Sibbett, W.

Sorrell, M. J.

M. J. Sorrell, J. Tribble, L. Reinisch, “Bacteria identification of otitis media with fluorescence spectroscopy,” Lasers Surg. Med. 14, 155–163 (1994);J. A. Werkhaven, L. Reinisch, M. J. Sorrell, J. Tribble, R. H. Ossoff, “Noninvasive optical diagnosis of bacteria causing otitis media,” Laryngoscope 104, 264–268 (1994);R. A. Dalterio, W. H. Nelson, D. Britt, J. F. Sperry, D. Psaras, J. F. Tanguay, S. L. Suib, “Steady-state and decay characteristics of protein tryptophan fluorescence from bacteria,” Appl. Spectrosc. 40, 86–90 (1986);B. V. Bronk, L. Reinisch, “Variability of steady-state bacterial fluorescence with respect to growth conditions,” Appl. Spectrosc. 47, 436–440 (1993);J. Ho, G. Fisher, “Detection of BW agents: flow cytometry measurement of Bacillus subtilis (BG) spore fluorescence,” Memo. 1421 (Defense Research Establishment, Suffield, Medicine Hat, Alberta, Canada, 1993).

Stetzenbach, L. D.

B. Lighthart, L. D. Stetzenbach, “Distribution of microbial bioaerosol,” in Atmospheric Microbial Aerosols, B. Lighthart, G. Mohr, eds. (Chapman & Hall, New York, 1994), Chap. 4, pp. 68–98.

Tribble, J.

M. J. Sorrell, J. Tribble, L. Reinisch, “Bacteria identification of otitis media with fluorescence spectroscopy,” Lasers Surg. Med. 14, 155–163 (1994);J. A. Werkhaven, L. Reinisch, M. J. Sorrell, J. Tribble, R. H. Ossoff, “Noninvasive optical diagnosis of bacteria causing otitis media,” Laryngoscope 104, 264–268 (1994);R. A. Dalterio, W. H. Nelson, D. Britt, J. F. Sperry, D. Psaras, J. F. Tanguay, S. L. Suib, “Steady-state and decay characteristics of protein tryptophan fluorescence from bacteria,” Appl. Spectrosc. 40, 86–90 (1986);B. V. Bronk, L. Reinisch, “Variability of steady-state bacterial fluorescence with respect to growth conditions,” Appl. Spectrosc. 47, 436–440 (1993);J. Ho, G. Fisher, “Detection of BW agents: flow cytometry measurement of Bacillus subtilis (BG) spore fluorescence,” Memo. 1421 (Defense Research Establishment, Suffield, Medicine Hat, Alberta, Canada, 1993).

Twichell, J. C.

R. K. Reich, R. W. Mountain, W. H. McGonagle, J. C. M. Huang, J. C. Twichell, B. B. Kosicki, E. D. Savoye, “Integrated electronic shutter for back-illuminated chargecoupled devices,” IEEE Trans. Electron Devices 40, 1231–1235 (1993).

Twomey, S.

S. Twomey, Atmospheric Aerosols (Elsevier, New York, 1977), Chap. 2, pp. 23–45.

Waggoner, A. P.

R. E. Weiss, A. P. Waggoner, R. J. Charlson, N. C. Ahlquist, “Sulfate aerosol: its geographical extent in the midwestern and southern United States,” Science 195, 979–981 (1977).

Weiss, R. E.

R. E. Weiss, A. P. Waggoner, R. J. Charlson, N. C. Ahlquist, “Sulfate aerosol: its geographical extent in the midwestern and southern United States,” Science 195, 979–981 (1977).

Young, K.

Zaruba, M. E.

R. C. Benson, R. A. Meyer, M. E. Zaruba, G. M. McKhann, “Cellular autofluorescence: is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).

Zoller, W.

J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).

Appl. Opt.

IEEE Trans. Electron Devices

R. K. Reich, R. W. Mountain, W. H. McGonagle, J. C. M. Huang, J. C. Twichell, B. B. Kosicki, E. D. Savoye, “Integrated electronic shutter for back-illuminated chargecoupled devices,” IEEE Trans. Electron Devices 40, 1231–1235 (1993).

J. Geophys. Res

J. M. Prospero, D. L. Savoie, R. T. Nees, R. A. Duce, J. Merrill, “Particulate sulfate and nitrate in the boundary layer over the north Pacific Ocean,” J. Geophys. Res. 90, 10,586–10,596 (1985).

J. Histochem. Cytochem

J. E. Aubin, “Autofluorescence of viable cultured mammalian cells,” J. Histochem. Cytochem. 27, 26–43 (1979).

R. C. Benson, R. A. Meyer, M. E. Zaruba, G. M. McKhann, “Cellular autofluorescence: is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).

J. Opt. Soc. Am

P. Chylek, “Partial-wave resonances and the ripple structure in the Mie normalized extinction cross section,” J. Opt. Soc. Am. 66, 285–287 (1976).

J. Opt. Soc. Am. A

P. Conwell, C. K. Rushforth, R. E. Benner, S. C. Hill, “Efficient automated algorithm for the sizing of dielectric microspheres using the resonance spectrum,”; J. Opt. Soc. Am. A 1, 1680–1687 (1984).

J. Opt. Soc. Am. B

Lasers Surg. Med.

M. J. Sorrell, J. Tribble, L. Reinisch, “Bacteria identification of otitis media with fluorescence spectroscopy,” Lasers Surg. Med. 14, 155–163 (1994);J. A. Werkhaven, L. Reinisch, M. J. Sorrell, J. Tribble, R. H. Ossoff, “Noninvasive optical diagnosis of bacteria causing otitis media,” Laryngoscope 104, 264–268 (1994);R. A. Dalterio, W. H. Nelson, D. Britt, J. F. Sperry, D. Psaras, J. F. Tanguay, S. L. Suib, “Steady-state and decay characteristics of protein tryptophan fluorescence from bacteria,” Appl. Spectrosc. 40, 86–90 (1986);B. V. Bronk, L. Reinisch, “Variability of steady-state bacterial fluorescence with respect to growth conditions,” Appl. Spectrosc. 47, 436–440 (1993);J. Ho, G. Fisher, “Detection of BW agents: flow cytometry measurement of Bacillus subtilis (BG) spore fluorescence,” Memo. 1421 (Defense Research Establishment, Suffield, Medicine Hat, Alberta, Canada, 1993).

Phys. Rev. Lett.

R. E. Benner, P. W. Barber, J. F. Owen, R. K. Chang, “Observation of structure resonances in the fluorescence spectra from microspheres,” Phys. Rev. Lett. 44, 475–479 (1980);A. J. Campillo, J. D. Eversole, H. B. Lin, “Cavity quantum electrodynamic enhancement of stimulated emission in microdroplets,” Phys. Rev. Lett. 67, 437–441 (1991);M. D. Barnes, W. B. Whitten, J. M. Ramsey, “Enhanced fluorescence yields through cavity-quantum electrodynamic effects in microspheres,” J. Chem. Phys. 97, 7842–7845 (1992).

Rev. Geophys. Space Phys.

J. M. Prospero, R. J. Charlson, V. Hohnen, R. Jaenicke, A. C. Delany, J. Moyers, W. Zoller, K. Rahn, “The atmospheric aerosol system: an overview,” Rev. Geophys. Space Phys. 21, 1607–1629 (1983).

Science

R. E. Weiss, A. P. Waggoner, R. J. Charlson, N. C. Ahlquist, “Sulfate aerosol: its geographical extent in the midwestern and southern United States,” Science 195, 979–981 (1977).

Other

S. Twomey, Atmospheric Aerosols (Elsevier, New York, 1977), Chap. 2, pp. 23–45.

B. Lighthart, L. D. Stetzenbach, “Distribution of microbial bioaerosol,” in Atmospheric Microbial Aerosols, B. Lighthart, G. Mohr, eds. (Chapman & Hall, New York, 1994), Chap. 4, pp. 68–98.

T. C. Eikhoff, “Perspectives on airborne infections in health care facilities,” in Proceedings of the Workshop on Engineering Controls for Preventing Airborne Infections in Workers in Health Care and Related Facilities, P. J. Bierbaum, M. Lippmann, eds. (National Institute of Occupational Safety and Health, Cincinnati, 1994), Pub. 94–106, pp. 15–34;Stockholm International Peace Research Institute, The Problem of Chemical and Biological Weapons, Vol. 1: The Rise of CB Weapons (Almqvist & Wiksell, Stockholm, 1971), pp. 111–124;U.S. Congress, Office of Technology Assessment, Technologies Underlying Weapons of Mass Destruction (U.S. GPO, Washington, D.C., 1993), Pub. OTA-BP-ISC-115, pp. 71–117.

R. G. Pinnick, S. C. Hill, P. Nachman, J. D. Pendleton, G. L. Fernandez, M. W. Mayo, J. G. Bruno, “Fluorescence particle counter for detecting airborne bacteria and other biological particles,” Aerosol Sci. Technol. (to be published).

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983), pp. 14–15.

Our conditional-sampling technique differs from familiar gating methods, such as boxcar integration. Conditional sampling operates on demand at the random times that interesting signals are present; in the usual gating methods, the detection systems are triggered in coincidence with phenomena that are also triggered.

P. Setlow, “Germination and outgrowth,” in The Bacterial Spore, A. Hurst, G. W. Gould, eds. (Academic, London, 1983), p. 214.

J. B. Perkins, J. G. Pero, “Biosynthesis of riboflavin, biotin, folic acid and cobalamin,” in Bacillus Subtilis and Other Gram Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, A. L. Sonensheim, J. A. Hoch, R. Losick, eds. (American Society for Microbiology, Washington, D.C., 1993), pp. 319–334.

We positioned the intracavity lens and the closure mirror so that, within the laser's gain medium, the extended cavity's circulating mode is the same as the original cavity's mode. The 25-cm focal length, plano–convex lens is made of fused silica. (We found that a comparable lens made of BK-7 glass, used in our initial study with the extended cavity, showed thermally induced distortion, caused by the high-power circulating intracavity. The fused-silica lens has much smaller thermal effects.) Wavelength selection is accomplished by the laser's intracavity prism and mode-defining aperture, still in their original locations. However, the wavelength discrimination is less effective in the modified configuration, so ~20% of the extended cavity's circulating power is actually in two additional laser lines (at 476.5 and 496.5 nm) adjacent to the dominant 488-nm line.

We did not use the PMS scattering cell-flow system intact because the light gathered by its specialized collection optics (a paraboloidal mirror segment covering more than 2π sr around the intersection volume of laser beam and aerosol stream) could not be focused to an image of sufficient quality for our purpose.

We were forced to collect light at 30° by our improvised flow system. This geometry may be suboptimal for detecting weak fluorescence in the presence of strong elastic scattering. However, because our setup was intracavity, we were also collecting light at 150° to the backward excitation beam; this may be a relatively favorable geometry for avoiding elastically scattered light. In any event, our spectra appear to be uncontaminated by elastic light.

The fiber-optics-coupled image-intensified CCD detector was from Princeton Instruments (Model ICCD-576ES). Its image intensifier was gated by a high-voltage pulser (Model FG-100). A controller (Model ST-130) read out and digitized signals from the CCD detector and sent them to our computer. The detector incorporates a CCD chip made by EEV, Ltd. (Chelmsford, Essex, U.K.) that has 22-μm-square pixels in an array that is 576 elements (horizontal) by 384 elements (vertical).

The fused-silica fiber has cladding, buffer, and jacket diameters of 660, 690, and 1200 μm, respectively. Its numerical aperture is 0.22. The fiber assembly was purchased from Polymicro Technologies, Inc., Phoenix, Ariz. 85023.

We also placed a long-pass (~500-nm) colored-glass filter just outside the spectrograph's entrance slit to attenuate the very bright elastically scattered light at 488 nm, thus minimizing stray light levels within the spectrograph and protecting the detector array's intensifier from possible damage. However, this filter transmitted enough at 488 nm to yield usable pulses in the elastic triggering channel.

AStanford Research DG535 digital delay generator is used in each channel to establish the voltage threshold for input triggering pulses and to set the time delay on the resulting transistor–transistor logic output pulses.

All spectra reported here were taken with an entrance slit width of 200 μm. The spectra were binned vertically (i.e., all the pixels in a vertical column of the CCD were summed to integrate all the captured light at each wavelength). The microchannel-plate intensifier was used with accelerating potentials between 800 and 900 V.

Duke Scientific Corp., 2463 Faber Place, Palo Alto, Calif. 94303.

The wavelength spacings of spectral peaks from the green-yellow fluorescing microspheres (4.5-μm diameter) are closer than the spacings from the pink-fluorescing spheres (1.96-μm diameter), because spacings decrease with particle size; see Ref. 27. Thus use of the two sphere types provides two independent confirmations of the conditional-sampling system's discrimination abilities, i.e., by means of peak spacings and by means of spectral region of fluoresence.

Likely noise sources in the PMT's are shot noise on their dark currents and on any dc-background light level. (The dc-background current would not have been evident to us, because the transimpedance amplifiers have ac-coupled outputs.)

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Figures (7)

Fig. 1
Fig. 1

Block diagram of the conditional-sampling spectrograph detection system. Colored-glass long-pass filters at the spectrograph entrance slit and before the fluorescence PMT are not shown.

Fig. 2
Fig. 2

Inelastic spectrum taken without conditional sampling. Although fluorescent microspheres were present, this 5-ms exposure in the free-running mode failed to capture spectra from any of them, recording only Raman scattering from atmospheric nitrogen and oxygen.

Fig. 3
Fig. 3

Fluorescence spectrum taken with conditional sampling. Elastic scattering from a 1.96-μm-diameter polystyrene microsphere doped with pink-fluorescing dye triggered a 50-μs exposure of the CCD detector, yielding the microsphere's fluorescence spectrum.

Fig. 4
Fig. 4

(a) Inelastic spectra taken without conditional sampling. Although 1.96-μm-diameter fluorescent microspheres (see Fig. 3) were present in the airstream, few of these 50-μs exposures taken in the free-running mode display particle fluorescence spectra. Raman scattering from atmospheric N2 is evident near 550 nm. (b) Same as (a), except taken with conditional sampling based on elastic scattering. Every frame in this set displays the fluorescence spectrum of an individual microsphere.

Fig. 5
Fig. 5

Fluorescence spectrum taken with conditional sampling. Elastic scattering from a 4.5-μm-diameter polystyrene microsphere doped with green- and yellow-fluorescing dyes triggered a 30-μs exposure of the CCD detector, yielding the microsphere's fluorescence spectrum. The voltage threshold was set high enough to avoid triggering on the 1.96-μm-diameter pink-fluorescing microspheres that were also present in the flow.

Fig. 6
Fig. 6

(a) Fluorescence spectra taken with conditional sampling. Fluorescence spectra of single 4.5-μm-diameter yellow-green fluorescent spheres (see Fig. 5) were reliably recorded in the presence of 1.96-μm-diameter pink fluorescent spheres by triggering from elastic scattering with the voltage threshold set high enough to exclude the smaller particles. Exposures were 30 μs. (b) Same as (a), except the voltage threshold was set low enough that elastic scattering from the smaller (1.96-μm-diameter) spheres was also able to trigger the system. (At this low threshold, noise spikes from the PMT may sometimes have triggered the system.) For clarity, intensities in frames showing spectra of the smaller spheres (shaded spectra) have been multiplied by two.

Fig. 7
Fig. 7

Bacterial fluorescence spectra taken with conditional sampling. Fluorescence spectra from single particles of Bacillus subtilis spores were recorded in the presence of nonfluorescing kaolin particles by triggering from total fluorescence of the bacteria. Each exposure was 200 μs. Thirty successive spectra from a set of 100 are shown here.

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