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

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.     

Simultaneous light scattering and intrinsic fluorescence measurement for the classification of airborne particles

We describe a prototype laboratory light-scattering instrument that integrates two approaches to airborne particle characterization: spatial light-scattering analysis and intrinsic fluorescence measurement, with the aim of providing an effective means of classifying biological particles within an ambient aerosol. The system uses a single continuous-wave 266-nm ultraviolet laser to generate both the spatial elastic scatter data (from which an assessment of particle size and shape is made) and the particle intrinsic fluorescence data from particles in the approximate size range of 1-10-mum diameter carried in a sample airflow through the laser beam. Preliminary results suggest that this multiparameter measurement approach can provide an effective means of classifying different particle types and can reduce occurrences of false-positive detection of biological aerosols.     

Fluorescence preselection of bioaerosol for single-particle mass spectrometry

We have designed, constructed, and tested a system that pre-selects the biological fraction of airborne particles from the overall aerosol. The preselection is based on fluorescence emission excited by a continuous 266 nm laser beam. This beam is one of two cw beams used to measure the aerodynamic particle size of sampled particles. The intention in our system is that single particles, based on size and fluorescence emission, can be selected and further examined for chemical composition by mass spectrometry.     

Single-particle mass spectrometry of polystyrene microspheres and diamond nanocrystals.

High-resolution mass spectra of single submicrometer-sized particles are obtained using an electrospray ionization source in combination with an audio frequency quadrupole ion-trap mass spectrometer. Distinct from conventional methods, light scattering from a continuous Ar-ion laser is detected for particles ejected out of the ion trap. Typically, 10 particles are being trapped and interrogated in each measurement. With the audio frequency ion trap operated in a mass-selective instability mode, analysis of the particles reveals that they all differ in mass-to-charge ratio (m/z), and the individual peak in the observed mass spectrum is essentially derived from one single particle. A histogram of the spectra acquired in 10(2) repetitions of the experiment is equivalent to the single spectrum that would be observed when an ion ensemble of 10(3) particles is analyzed simultaneously using the single-particle mass spectrometer (SPMS). To calibrate such single-particle mass spectra, secular frequencies of the oscillatory motions of the individual particle within the trap are measured, and the trap parameter qz at the point of ejection is determined. A mass resolution exceeding 10(4) can readily be achieved in the absence of ion ensemble effect. We demonstrate in this work that the SPMS not only allows investigations of monodisperse polystyrene microspheres, but also is capable of detecting diamond nanoparticles with a nominal diameter of 100 nm, as well.     

Scattering and Internal Fields of a Microsphere that Contains a Saturable Absorber: Finite-Difference Time-Domain Simulations

Illumination intensities that are used to induce scattering and fluorescence in aerosols can be large enough to cause variations in the refractive index. Methods used to calculate the scattering from homogeneous particles may not be valid for these systems. We use the finite-difference time-domain method and an iterative technique to model scattering by microspheres that contain a saturable absorber. We illustrate this technique by calculating the scattering from spheres that contain tryptophan. We show the Mueller scattering matrices along with the internal intensity distributions for different incident intensities. The backscattering increases as the illumination intensity becomes large enough to saturate the absorption in regions of the sphere.     

Influence of saline media on the fluorescence emission of Bacillus spores

Single-particle levitation in conjunction with 264.3-nm laser excitation is used to measure the fluorescence emission of individual particles of Bacillus globigii spores. With precise humidity control, the fluorescence emission of wetted and desiccated Bacillus spore particles is measured from 300 to 450 nm. Comparison of spectra for Bacillus spores suspended in a standard buffer aqueous solution and for a desiccated 10-mum-diameter aggregate Bacillus spore particle shows that the spectra is virtually indistinguishable. However, at 85% relative humidity, corresponding to a 4.5M sodium chloride solution, the spore spectra redshifts by approximately 25 nm. It is postulated that the spectra redshifting is a result of specific interactions between the tyrosine fluorophore of the Bacillus spore and the phosphate moieties in the buffer solution.     

Desorption/ionization fluence thresholds and improved mass spectral consistency measured using a flattop laser profile in the bioaerosol mass spectrometry of single Bacillus endospores

Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time. Mass spectra of individual Bacillus endospores were measured with a bipolar aerosol time-of-flight mass spectrometer in which molecular desorption and ionization were produced using a single laser pulse from a Q-switched, frequency-quadrupled Nd:YAG laser that was modified to have an approximately flattop profile. The flattened laser profile allowed the minimum fluence required to desorb and ionize significant numbers of ions from single aerosol particles to be determined. For Bacillus spores, this threshold had a mean value of approximately 1 nJ/microm(2) (0.1 J/cm(2)). Thresholds for individual spores, however, could apparently deviate by 20% or more from the mean. Threshold distributions for clumps of MS2 bacteriophage and bovine serum albumin were subsequently determined. Finally, the flattened profile was observed to increase the reproducibility of single-spore mass spectra. This is consistent with the general conclusions of our earlier paper on the fluence dependence of single-spore mass spectra and is particularly significant because it is expected to enable more robust differentiation and identification of single bioaerosol particles.     

Asbestos real-time monitor in an atmospheric environment

The concentration of asbestos fiber aerosols can be monitored by measuring the polarization of laser light scattered by asbestos fibers. The principle of discriminating asbestos fibers is based on the theoretically expected difference in polarization at a scattering angle of 170 deg between cylindrical and spherical airborne particles; polarization at this scattering angle should be positive for cylindrical particles such as asbestos fibers but should be negative or close to zero for spherical mineral particles. We constructed an experimental asbestos real-time monitor that uses a strong electric field to align the airborne particles, that uses lasers having linear polarization with an equal amplitude in parallel and perpendicular components to the aligned long axis of particles, and that simultaneously detects the two components of the linear polarization of light scattered at 170 deg, i.e., close to the backscatter. Experiments that were performed to detect the light scattered from airborne standard asbestos fibers showed that the measured polarization fits theoretical prediction. The concentrations of airborne asbestos fibers obtained by the asbestos real-time monitor were consistent with those estimated by the standard phase contrast microscope method.     

Surface-enhanced Raman spectroscopy of bacteria and pollen

A technique for distinguishing biological material based on surface-enhanced Raman scattering (SERS) is reported in this work. Of particular interest is biological material that can be airborne. Silver colloidal particles with diameters in the range 10 to 20 nm and with a characteristic ultraviolet-visible (UV-VIS) absorption band at 400 nm were used to obtain SERS spectra of Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium bacteria and a number of tree and grass pollens (Cupressus arizonica (cypress), Sequoia sempervirens (redwood), Populus deltoides (cottonwood), Poa pratensis (Kentucky bluegrass), and Anthoxanthum odoratum (sweet vernal grass)). While differences in the SERS spectra among the bacteria were small, we found that the pollen spectra we analyzed could readily be distinguished from the bacteria spectra, and there were significant differences between pollen from different families. In order to obtain reproducible results, we studied the parameters controlling the interaction between the analyte and the nanoscale metallic surface. Our results show that the volume ratio of analyte to colloidal particles must be within a narrow range of values to optimize the signal-to-noise ratio of the SERS spectra and minimize the fluorescence from the analyte. Also, we found that the time-dependent behavior of colloidal/bacterial suspensions (or adsorption rate of the silver colloid particles on the bacteria) is strongly dependent on pH, density of bacteria in solution, and even, to some extent, the type of bacteria.     

Discrimination between bacterial spore types using time-of-flight mass spectrometry and matrix-free infrared laser desorption and ionization

We demonstrate that molecular ions with mass-to-charge ratios (m/z) ranging from a few hundred to 19 050 can be desorbed from whole bacterial spores using infrared laser desorption and no chemical matrix. We have measured the mass of these ions using time-of-flight mass spectrometry and we observe that different ions are desorbed from spores of Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Bacillus niger. Our results raise the possibility of identifying microorganisms using mass spectrometry without conventional sample preparation techniques such as the addition of a matrix. We have measured the dependence of the ion yield from B. subtilis on desorption wavelength over the range 3.05-3.8 microm, and we observe the best results at 3.05 microm. We have also generated mass spectra from whole spores using 337-nm ultraviolet laser desorption, and we find that these spectra are inferior to spectra generated with infrared desorption. Since aerosol analysis is a natural application for matrix-free desorption, we have measured mass spectra from materials such as ragweed pollen and road dust that are likely to form a background to microbial aerosols. We find that these materials are readily differentiated from bacterial spores.     

Backward-enhanced fluorescence from clusters of microspheres and particles of tryptophan

Measured fluorescence from single-particle clusters of dye-doped polystyrene microspheres, dried nonspherical particles of tryptophan, and single polystyrene microspheres is enhanced in the backward direction (180 degrees from the incident laser). This enhancement (a factor of 2-3 compared to 90 degrees), which can be interpreted as a consequence of the reciprocity principle, increases with the particle refractive index.     

Principal-components analysis of fluorescence cross-section spectra from pathogenic and simulant bacteria

Principal-components analysis of a new set of highly resolved (< 1 nm) fluorescence cross-section spectra excited at 354.7 nm over the 370-646 nm band has been used to demonstrate the potential ability of UV standoff lidars to discriminate among particular biological warfare agents and simulants over short ranges. The remapped spectra produced by this technique from Bacillus globigii (Bg) and Bacillus anthracis (Ba) spores were sufficiently different to allow them to be cleanly separated, and the Ba spectra obtained from Sterne and Ames strain spores were distinguishable. These patterns persisted as the spectral resolution was subsequently degraded in processing from approximately 1 to 34 nm. This is to the author's knowledge the first time that resolved fluorescence spectra from biological warfare agents have been speciated or shown to be distinguishably different from those normally used surrogates by optical spectroscopy.     

微米级ZnS-Qds@聚硅氧烷核-壳型光扩散杂化微球的制备与应用

在液晶背光模组中通常使用扩散膜来提高光的散射效果,提高光的利用率。光散射粒子是影响光散射材料透光率和雾度的主要因素。有机硅微球由于热稳定性好,尺寸稳定性佳,粒径和折射率可以通过反应调控的特点,作为光扩散粒子应用于光扩散材料具有十分广阔的应用前景。本文通过在溶胶-凝胶法制备的聚硅氧烷微球上面沉淀一层被巯基聚硅氧烷包裹的硫化锌量子点(ZnS-Qds),制得微米级ZnS-Qds@聚硅氧烷核-壳型光扩散杂化微球,并制备了添加不同质量分数的杂化微球的光扩散膜。采用TEM、TEM映射分析、荧光光度计等测试手段对所制备的杂化材料进行了表征,并对用该杂化粒子制备得到的光扩散膜进行了光学性能测试。结果表明,ZnS-Qds@聚硅氧烷光扩散杂化微球具有核壳结构,平均粒径约3.6μm,壳层平均厚度为87.4 nm,平均粒径为2 nm的ZnS-Qds均匀地分散在壳层中。将该粒子添加到丙烯酸树脂中并涂覆在聚对苯二甲酸乙二醇酯(PET)膜上以制成光扩散膜,实验结果表明,当杂化粒子添加量从0wt%增加到20wt%时,光扩散膜的雾度从1.67%提高到91.11%,而光扩散膜的透光率仅从90.10%降低到82.57%。     

Beam attenuation coefficient retrieval by inversion of airborne lidar-induced chromophoric dissolved organic matter fluorescence. I. Theory

It is shown that the oceanic beam attenuation coefficient can be retrieved from airborne laser-induced and depth-resolved chromophoric dissolved organic matter (CDOM) fluorescence. The radiative transfer equation (RTE) retrieval methodology does not require a laser beam spread function model since two CDOM fluorescence bands are used in conjunction with a beam attenuation spectral model, is self-normalizing since the CDOM absorption coefficient and laser beam irradiance are common to both fluorescence observational channels, and is enabled by the known isotropic phase function for CDOM fluorescence. Although this RTE analytical inversion theory is exact, the retrieval uncertainty is reduced by configuring the proposed lidar in the multiple-field-of-view beam attenuation mode to significantly diminish observation of multiple scattering. The theory can be applied over wide regions of the ocean's continental margins, estuaries, lakes, and rivers that are known to have sufficient CDOM.     

Laser-induced breakdown spectroscopy detection and classification of biological aerosols

Laser-induced breakdown spectroscopy (LIBS) is examined as a potential method for detecting airborne biological agents. A spectrally broadband LIBS system was used for laboratory measurements on some common biological agent simulants. These measurements were compared to those of common, naturally occurring biological aerosol components (pollen and fungal spores) to determine the potential of LIBS for discriminating biological agents from natural background aerosols. A principal components analysis illustrates that linear combinations of the detected atomic lines, which are present in different ratios in each of the samples tested, can be used to discriminate biological agent simulants from other biological matter. A more sensitive, narrowband LIBS instrument was used to demonstrate the detection of single simulant (Bg) particles in the size range 1-5 microns. Ca, Mg, and Na, which are present in varying concentrations between 0.3 and 11% (by mass) in the Bg particles, were observed in single particles using LIBS.     

Optical trapping of fluorescent beads

Optical tweezers have been successfully used to trap a variety of particles and biological specimens for numerous applications. Particles which are reflective as well as absorbing could be trapped using beams such as optical vortex. Here we give the details of our efforts to trap fluorescent microparticles. We have set up an optical trap for these fluorescent microparticles using holographic optical tweezers; we observe that it is not possible to trap fluorescent microparticles with a Gaussian laser beam or a hollow beam. However, as the fluorescence of these particles gets degraded they could be trapped in custom-made holographic tweezers. Moreover, when a fluorescent particle is brought in the trap containing stably trapped non-fluorescent particle, the stably trapped non-fluorescent particle also escapes from the trap.     

Two-dimensional multiwavelength fluorescence spectra of dipicolinic acid and calcium dipicolinate

Dipicolinic acid (DPA) and the Ca2+ complex of DPA (CaDPA) are major chemical components of bacterial spores. With fluorescence being considered for the detection and identification of spores, it is important to understand the optical properties of the major components of the spores. We report in some detail on the room-temperature fluorescence excitation and emission spectra of DPA and its calcium ion complex and provide a comparison of the excitation-emission spectrum in a dry, wet paste and aqueous form. DPA solutions have weak, if any, fluorescence, with increased fluorescence when the DPA is dry. After exposure to a broad source UV light of the DPA, wet or dry, we observe a large increase in fluorescence with a maximum intensity emission peak at around 440 nm for excitation light with a wavelength of around 360 nm. There is a slight blueshift in the absorption spectra of UV-exposed DPA from the unexposed DPA solution. CaDPA in solution shows a slight fluorescence with increased fluorescence in the dry form, and a substantial increase of fluorescence was observed after UV exposure with an emission peak of around 410 nm for excitation around 305 nm. The detailed excitation-emission spectra are necessary for better interpretation of the fluorescence spectra of bacterial spores where DPA is a major chemical component.     

Cross-type optical particle separation in a microchannel

A continuous, real-time optical particle separation, which was previously delineated theoretically, is successfully implemented experimentally for the first time. In this method, particles suspended in a flowing fluid are irradiated with a laser beam propagating in a direction perpendicular to direction of fluid flow. Upstream of the laser beam, the particles move parallel to the direction of fluid flow. When the particles pass through the laser beam, the scattering force pushes them in the direction of laser beam propagation, causing the particles to be displaced perpendicular to the fluid flow direction. This displacement, known as the retention distance, depends on the particle size and the laser beam parameters. Finally, the particles escape from the laser beam and maintain their retention distances as they move downstream. In the present work, the trajectories and retention distances of polystyrene latex microspheres with three distinct diameters were monitored and measured using cross-type optical particle separation. The measured retention distances for different-sized particles were in good agreement with theoretical predictions.     

Effect of washing on identification of Bacillus spores by principal-component analysis of fluorescence data

The fluorescence spectra of Bacillus spores are measured at excitation wavelengths of 280, 310, 340, 370, and 400 nm. When cluster analysis is used with the principal-component analysis, the Bacillus globigii spores can be distinguished from the other species of Bacillus spores (B. cereus, B. popilliae, and B. thuringiensis). To test how robust the identification process is with the fluorescence spectra, the B. globigii is obtained from three separate preparations in different laboratories. Furthermore the fluorescence is measured before and after washing and redrying the B. globigii spores. Using the cluster analysis of the first two or three principal components of the fluorescence spectra, one is able to distinguish B. globigii spores from the other species, independent of preparing or washing the spores.     

Photodeposition of silver can result in metal-enhanced fluorescence

Chemically deposited silver particles are widely used for surface-enhanced Raman scattering (SERS) and more recently for surface-enhanced fluorescence (SEF), also known as metal-enhanced fluorescence (MEF). We now show that metallic silver deposited by laser illumination results in an approximately 7-fold increased intensity of locally bound indocyanine green. The increased intensity is accompanied by a decreased lifetime and increased photostability. These results demonstrate the possibility of photolithographic preparation of surfaces for enhanced fluorescence in microfluidics, medical diagnostics, and other applications.