Aerosol-fluorescence spectrum analyzer: real-time measurement of emission spectra of airborne biological particles

We have assembled an aerosol-fluorescence spectrum analyzer (AFS), which can measure the fluorescence spectra and elastic scattering of airborne particles as they flow through a laser beam. The aerosols traverse a scattering cell where they are illuminated with intense (50 kW/cm(2)) light inside the cavity of an argon-ion laser operating at 488 nm. This AFS can obtain fluorescence spectra of individual dye-doped polystyrene microspheres as small as 0.5 μm in diameter. The spectra obtained from microspheres doped with pink and green-yellow dyes are clearly different. We have also detected the fluorescence spectra of airborne particles (although not single particles) made from various biological materials, e.g., Bacillus subtilis spores, B. anthrasis spores, riboflavin, and tree leaves. The AFS may be useful in detecting and characterizing airborne bacteria and other airborne particles of biological origin.     

Reagentless identification of single bacterial spores in aqueous solution by confocal laser tweezers Raman spectroscopy

We demonstrate that optical trapping combined with confocal Raman spectroscopy using a single laser source is a powerful tool for the rapid identification of micrometer-sized particles in an aqueous environment. Optical trapping immobilizes the particle while maintaining it in the center of the laser beam path and within the laser focus, thus maximizing the collection of its Raman signals. The single particle is completely isolated from other particles and substrate surfaces, therefore eliminating any unwanted background signals and ensuring that information is collected only from the selected, individual particle. In this work, an inverted confocal Raman microscope is combined with optical trapping to probe and analyze bacterial spores in solution. Rapid, reagentless detection and identification of bacterial spores with no false positives from a complex mixed sample containing polystyrene and silica beads in aqueous suspension is demonstrated. In addition, the technique is used to analyze the relative concentration of each type of particle in the mixture. Our results show the feasibility for incorporating this technique in combination with a flow cytometric-type scheme in which the intrinsic Raman signatures of the particles are used instead of or in addition to fluorescent labels to identify cells, bacteria, and particles in a wide range of applications.     

Discrimination between Bacillus species by impedance analysis of individual dielectrophoretically positioned spores

We combine the use of dielectrophoretic positioning with electrical impedance measurements to detect and discriminate between individual bacterial spores on the basis of their electrical response. Using lithographically defined microelectrodes, we use dielectrophoresis to manipulate individual bacterial spores between the electrodes. The introduction of a single spore between the microelectrodes produces a significant change in electrical response that is species-dependent. When positioned between two electrodes and an AC voltage was applied, single spores caused current increases averaging 6.8 (+/-2.4) pA for Bacillus mycoides to 1.18 (+/-0.37) pA for Bacillus licheniformis. Using a mixture of spores of two different species, we demonstrate the ability to distinguish the species of individual spores in real time. This work demonstrates the feasibility of using impedance measurements for real-time detection and discrimination between different types of spores.     

Simultaneous laser-induced fluorescence and scattering detection of individual particles separated by capillary electrophoresis

This technical note describes a detector capable of simultaneously monitoring scattering and fluorescence signals of individual particles separated by capillary electrophoresis. Due to its nonselective nature, scattering alone is not sufficient to identify analyte particles. However, when the analyte particles are fluorescent, the detector described here is able to identify simultaneously occurring scattering and fluorescent signals, even when contaminating particles (i.e., nonfluorescent) are present. Both fluorescent polystyrene particles and 10-nonyl acridine orange (NAO)-labeled mitochondria were used as models. Fluorescence versus scattering (FVS) plots made it possible to identify two types of particles and a contaminant in a mixture of polystyrene particles. We also analyzed NAO-labeled mitochondria before and after cryogenic storage; the mitochondria FVS plots changed with storage, which suggests that the detector reported here is suitable for monitoring subtle changes in mitochondrial morphology that would not be revealed by monitoring only fluorescence or scattering signals.     

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.     

Laser power dependence of mass spectral signatures from individual bacterial spores in bioaerosol mass spectrometry

Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time. Characteristic mass spectra from individual bacterial endospores of Bacillus subtilis var. niger were obtained in a bipolar aerosol time-of-flight mass spectrometer using a pulsed 266-nm laser for molecular desorption and ionization. Spectra from single spores collected at an average fluence of approximately 0.1 J/cm2 frequently contain prominent peaks attributed to arginine, dipicolinic acid, and glutamic acid, but the shot-to-shot (spore-to-spore) variability in the data may make it difficult to consistently distinguish closely related Bacillus species with an automated routine. Fortunately, a study of the laser power dependence of the mass spectra reveals clear trends and a finite number of "spectral types" that span most of the variability. This, we will show, indicates that a significant fraction of the variability must be attributed to fluence variations in the profile of the laser beam.     

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.     

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.     

羧基功能化聚苯乙烯荧光微球的制备及表征

以苯乙烯、丙烯酸为单体,引入疏水性荧光染料罗丹明6G(Rh6G),采用微乳聚合法制备羧基聚苯乙烯荧光微球,并分析了表面活性剂、引发剂、丙烯酸用量对产物粒径分布的影响,考察了羧基聚苯乙烯荧光微球的浓度对荧光强度的影响。通过粒度分析仪、扫描电子显微镜、红外光谱仪、紫外吸收光谱以及荧光光谱仪对样品的纳米特性、形貌、结构和荧光性能进行了表征。结果表明,用微乳聚合法制备出50~250nm的羧基聚苯乙烯荧光微球,粒径均一且呈单分散性。紫外光谱图测试表明,在533nm左右有吸收峰。荧光光谱测试表明,羧基功能化的荧光聚苯乙烯微球浓度≤0.01%,其荧光最大激发峰为527nm,最大发射峰在555nm处;浓度高于0.01%时,荧光光谱出现红移,且荧光强度减弱。     

Development of size-selective sampling of Bacillus anthracis surrogate spores from simulated building air intake mixtures for analysis via laser-induced breakdown spectroscopy

Size-selective sampling of Bacillus anthracis surrogate spores from realistic, common aerosol mixtures was developed for analysis by laser-induced breakdown spectroscopy (LIBS). A two-stage impactor was found to be the preferential sampling technique for LIBS analysis because it was able to concentrate the spores in the mixtures while decreasing the collection of potentially interfering aerosols. Three common spore/aerosol scenarios were evaluated, diesel truck exhaust (to simulate a truck running outside of a building air intake), urban outdoor aerosol (to simulate common building air), and finally a protein aerosol (to simulate either an agent mixture (ricin/anthrax) or a contaminated anthrax sample). Two statistical methods, linear correlation and principal component analysis, were assessed for differentiation of surrogate spore spectra from other common aerosols. Criteria for determining percentages of false positives and false negatives via correlation analysis were evaluated. A single laser shot analysis of approximately 4 percent of the spores in a mixture of 0.75 m(3) urban outdoor air doped with approximately 1.1 x 10(5) spores resulted in a 0.04 proportion of false negatives. For that same sample volume of urban air without spores, the proportion of false positives was 0.08.     

Spectrally resolved absolute fluorescence cross sections for bacillus spores

Absolute fluorescence cross sections for Bacillus subtilis and B. cereus bacterial spores as both aqueous suspensions and aerosols were measured at a number of excitation wavelengths between 228 and 303 nm. The fluorescence was spectrally resolved at each excitation wavelength. We found that the optimum excitation wavelength for spore fluorescence is between 270 and 280 nm. The fluorescence cross section for aqueous suspensions is four times larger than for dry aerosols when measured under similar conditions. Measurements on wet aerosols showed an increase in fluorescence cross section over dry aerosols, indicating an enhancement of the fluorescence when the bacterial spores are wet. Mie scattering cross sections at 90 degrees to the direction of the incident radiation and extinction cross sections as a function of wavelength for B. subtilis suspensions and fluorescence cross sections for tryptophan are also reported.     

Modeling biological fluorescence emission spectra using Lorentz line shapes and nonlinear optimization

Using the Levenberg-Marquardt nonlinear optimization algorithm and a series of Lorentzian line shapes, the fluorescence emission spectra from BG (Bacillus globigii) bacteria can be accurately modeled. This method allows data from both laboratory and field sources to model the return signal from biological aerosols using a typical LIF (lidar induced fluorescence) system. The variables found through this procedure match individual fluorescence components within the biological material and therefore have a physically meaningful interpretation. The use of this method also removes the need to calculate phase angles needed in autoregressive all-pole models.     

X-ray CCD calibration system using fluorescent lines

We have developed a CCD calibration system using fluorescent X-ray lines with energies ranging from 1.49 keV (Al K) to 11.2 keV (Se K). The absolute X-ray flux is calibrated by a gas proportional counter, while the emerging spectra are monitored by solid-state silicon detectors. In order to suppress contaminating X-rays in the fluorescence spectra, mechanical collimators were set in the X-ray beam line, high-purity targets for fluorescent lines were used, and band-pass filters were put on the X-ray beam line. As for the purity of the fluorescent X-rays, the typical purity achieved was 98%.     

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.     

Multiplexed spectral signature detection for microfluidic color-coded bioparticle flow

Here, we report a high-speed photospectral detection technique capable of discriminating subtle variations of spectral signature among fluorescently labeled cells and microspheres flowing in a microfluidic channel. The key component used in our study is a strain-tunable nanoimprinted grating microdevice coupled with a photomultiplier tube (PMT). The microdevice permits acquisition of the continuous spectral profiles of multiple fluorescent emission sources at 1 kHz. Optically connected to a microfluidic flow chamber via a multimode optical fiber, our multiwavelength detection platform allows for cytometric measurement of cell groups emitting nearly identical fluorescence signals with a maximum emission wavelength difference as small as 5 nm. The same platform also allows us to demonstrate microfluidic flow cytometry of four different microsphere types in a wavelength bandwidth as narrow as 40 nm at a high (>85%) confidence level. Our study shows that detection of fluorescent spectral signatures at high speed and high resolution can expand specificity of multicolor flow cytometry. The enhanced capability enables multiplexed analysis of color-coded bioparticles based on single-laser excitation and single-detector spectroscopy in a microfluidic setting. The fluorescence signal discrimination power achieved by the optofluidic technology holds great promise to enable quantification of cellular parameters with higher accuracy as well as enumeration of a larger number of cell types than conventional flow cytometric methods.     

Spatial fluorescence cross-correlation spectroscopy

We present an alternative method for diffusion measurements of fluorescent species in solution by use of confocal microscopy and fluorescence correlation spectroscopy techniques. It consists of making a time and spatial dual correlation in which one detects the fluorescence signals from two nearby separate confocal volumes and cross correlates them. To improve the spatial discrimination between the two confocal volumes we propose filtering of fluorescence photocounts by rejecting the fluorescence background, which corresponds to particles located far from the center of the detection volumes.     

Resolution of multicomponent fluorescence emission using frequency-dependent phase angle and modulation spectra

We describe a new fluorescence method that allows the resolution of both the decay times and emission spectra of mixtures of fluorophores. This method is completely general and does not require any assumptions or knowledge of the decay times or emission spectra of the individual fluorophores. We use the phase angle spectra and modulation spectra of the mixture, measured over a range of suitable light modulation frequencies and emission wavelengths. These data are analyzed by nonlinear least-squares analysis to recover the emission spectra and the associated decay times. The principle of the method and the nature of the data are illustrated by using two-component mixtures with increasing spectral overlap. We then demonstrate the recovery of minor components, of structure emission spectra, and of a three-component mixture with completed overlapping emission spectra. And finally, we describe the resolution of a two-component mixture with decay times of 0.8 and 1.4 ns using modulation frequencies up to 774 MHz.     

Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy

Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small dye molecule. It was found that bacteria and microspheres are retarded in their flow by optical forces produced by the laser beam.     

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.