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Applied Optics

Applied Optics


  • Vol. 35, Iss. 7 — Mar. 1, 1996
  • pp: 1069–1076

Conditional-sampling spectrograph detection system for fluorescence measurements of individual airborne biological particles

Paul Nachman, Gang Chen, R. G. Pinnick, Steven C. Hill, Richard K. Chang, Michael W. Mayo, and Gilbert L. Fernandez  »View Author Affiliations

Applied Optics, Vol. 35, Issue 7, pp. 1069-1076 (1996)

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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

Original Manuscript: May 22, 1995
Revised Manuscript: August 8, 1995
Published: March 1, 1996

Paul Nachman, Gang Chen, R. G. Pinnick, Steven C. Hill, Richard K. Chang, Michael W. Mayo, and Gilbert L. Fernandez, "Conditional-sampling spectrograph detection system for fluorescence measurements of individual airborne biological particles," Appl. Opt. 35, 1069-1076 (1996)

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  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.
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  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.)
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