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R. A. FLETCHER, J. A. SMALL, and J. H. J. SCOTT National Institute of Standards and Technology, Gaithersburg, MD
INTRODUCTION Important information about an aerosol resides in the morphology and in the chemical composition of the individual particles. Chemical, morphological, phase, and crystallographic data are often crucial for purposes of tracing formation mechanisms and possible sources of airborne particulate matter. Applications of single-particle analysis include clean room technology, microelectronics, indoor and outdoor air pollution, forensics, and many problems related to microcontamination. Analytical instruments and techniques must be employed to obtain compositional information at the single-particle level. Digital image processing plays an increased role in providing new information about chemical composition and morphological properties of microstructures and particles. Single-particle analysis is defined here to mean the analysis of collected, individual particles ranging from about 5nm to several millimeters in lateral dimension. The information obtained includes the chemical constituents that make up the particle, the morphology (size, shape), and the physical and optical properties. Two requirements must be met for singleparticle analysis: The analytical technique must have sufficient spatial resolution to differentiate a single particle from the background substrate material and adjacent particles and sufficient sensitivity to detect at least major components of the particle's composition (picogram detection levels for micrometer-sized unit density particles). Microanalysis, entailing the use of microscopes or microprobes, often fulfills these requirements. Microanalysis by means of microscopy originated with the advent of the light microscope, while the first microprobe work resulted from the invention of the electron microprobe (Castaing and Guinier, 1950). Microanalytical developments have been motivated by the need to obtain images, elemental and molecular (compound) information on a microscopic scale. In microanalysis, a beam of excitation radiation, which can be photons, electrons, protons, neutrons, or ions, is used to bombard the sample. The interaction of the beam with the sample results in emitted radiation that is separated by a spectrometer and collected by a detector, as shown in Figure 12-1. For microprobes, the excitation beam is focused on the particle with a spatial resolution ranging from nanometers to micrometers. To create an image, either the excitation beam is rastered across the particle or the mounted particle is translated under the beam. Microscopes usually flood the sample with excitation radiation, but form an image by focusing the secondary radiation. Spatially resolved analysis is achieved in the microscope either
Aerosol Measurement: Principles, Techniques, and Applications, Second Edition, Edited by Paul A. Baron and Klaus Willeke. ISBN 0-471-35636-0 Copyright © 2001 Wiley-InterScience, Inc.
where the detected beam is frequency-shifted photons. The emissions are photons in the infrared. The instruments discussed are the light microscope. For quick reference. Some specific examples of instruments using electron beam excitation are electron probes. and analytical electron microscopes. which is based on photon absorption. the laser microprobe. and morphological information) about that particle (Steel et al. laser. This chapter describes microscopes and microprobes used for analysis of collected. an argon. and the review chapter by Grasserbauer (1978). Schematic representation of generic microanalysis showing excitation and emission radiation. or X-rays. chemical. which detects ions in a time-of-flight mass spectrometer. is used for single-particle analysis. complementary capabilities of two or more instruments can be used in sequence on an individual particle to provide more complete particle characterization (phase. ultraviolet. 1984. The chapter also contains some basic information about sample preparation. Examples of instruments that use ion beams are the ion microprobe and microscope based on secondary ion mass spectrometry (SIMS). the article by Newbury (1990). backscattered electrons or X-rays. . ions. oxygen.source primary radiation (ens secondary radiation spectrometer specimen detector Fig. and the Fourier transform infrared microscope.1999). Several excellent references that should be consulted for more detailed information include the books by Spurny (1986. electron microprobes. Often in an analysis. by masking a portion of the secondary radiation (aperturing) or by employing a spatially sensitive detector.. individual particles. Examples of instruments that use photon radiation are the Raman microprobe (or micro-Raman). The principles of operation and the instrumental capabilities are presented. electron microscopes (both scanning and transmission). Table 12-1 presents a comparison of the microanalytical techniques capable of single-particle analysis. and ion microprobes. scanning probe. useful for the aerosol scientist. 12-1. scanning electron microscopes. optical. photons. The emitted radiation. Fletcher et al. 1990).. or cesium ion beam is used to bombard the sample. In SIMS. the publication by Heinrich (1980). and either a time-of-flight mass spectrometer or more usually a magnetic sector or quadrupole mass spectrometer is used to detect the secondary ions generated in the interaction process. or ions. or visible. The incident radiation can be electrons.
(1980).A. N. N.A. . X-rays 0. X-rays 5nm >Carbon No None 10"16g 0.A.1 um (probe) AU Yes Organic and inorganic io-19g 1-1000 ppm Yes Yes Yes Yes No No N.A.A. N. X-rays 0.A.5 um N.1% Yes No No Yes Yes Yes TEM Electrons Electrons. No No No Yes Yes No FT-IR Photons Photons Intensity at given wavelength 10-50 inm N.3 nm >Carbon No Inorganic io-2Og 0. Source: Adapted from Wieser et al. N. N.1% Yes No No Yes Yes Yes LMMS Photons Ions m/z lum AU Yes Organic and inorganic 10"18 to 10-20g 1-100 ppm Yes Yes Unknown Demonstrated. Organic and inorganic 5 x l(T12g 1% No No No Yes Yes Semi AFM N. Organic and inorganic 10"12g 1% No No No Yes Yes No N.1% Yes No No Yes Yes Yes EPMA Electrons Electrons X-rays Electrons. not widely available No No a SIMS Ions Ions m/z Micro-Raman Photons Photons Frequency shift 2um LM Photons Photons Light intensity 0.A.TABLE 12-1. Inorganic N.A. Automation demonstrated Quantitative a Photons in the UV to IR range. X-rays Electrons. N. N. force. N.A. X-rays Electrons.A. other interactions 0. Distance.A. Comparison of Typical Microbeam Analytical Instrument Capabilities Analytical Method Excitation Emission Quantity measured Lateral resolution Detectable elements Isotopic detection Detectability of molecular or chemical compounds Absolute detection sensitivity Relative sensitivity Sample vacuum mounted Destructive method Surface sensitive Imaging capability SEM Electrons Electrons.A.A. N.05 um >Carbon No None io-16g 0.A.A.A. No No Yes Yes Yes N.
and resolving power. There are a number of optical accessories used in conjunction with the light microscope to characterize a sample physically. The image is focused on the detector. Table 12-2 presents characteristics of various common microscope objectives (Steel.3% of the incident light to be visible to the eye (Dovichi and Burgi. Light is transmitted through the sample and focused by an objective lens. numerical aperture. Normally. and an ocular. An object must absorb approximately 0. Eye or Camera Ocular Lens Image Objective Lens Object Virtual Image Fig. The light source can be diffuse or bright and serves to illuminate the sample. The ocular magnifies the image that is projected by the objective for the eye. The intermediate image is then enlarged and transmitted to the eye or the detector. Instrumentation A schematic of a light microscope is presented in Figure 12-2. the light microscope utilizes light refraction via a lens system to form enlarged images of microscopic objects. the virtual image seen resides below the sample plane. The objective lens collects light that passes through the sample or is reflected from the sample surface and projects an image near the ocular. The operating principle of light microscopes is well known. Schematic of a light microscope.) . referring to the distance beyond the plane of focus that the object remains in focus. 12-2. relating the maximum light-gathering capability of the microscope objective. quantifying the image enlargement. 1980). The important components of a light microscope are a light source.LIGHT MICROSCOPY Underlying Principle Optical microscopy or light microscopy (LM) is one of the more familiar microanalytical techniques. indicating the size of the smallest feature that can be discriminated. (Adapted from McCrone and Delly. magnification. an objective lens. In the simplest form. which can be the human eye or a camera. Some considerations in LM are depth of field. Some of these capabilities are discussed later. 1987). 1973.
but some additional physical properties help to provide more definitive information about a particle's makeup. A skilled microscopist can use the physical and optical properties of a particle (such as the size. Source: Steel (1980). particle magnification in the light microscope is limited due to the wavelength of light used (diffraction limit).5 um size level.3 Depth of Field (M-m) 50 8 1 0. Fibers may be asbestos or glass or may come from a wide variety of other natural or man-made sources. The Particle Atlas (McCrone and Delly.TABLE 12-2. 1973) is regarded as a principal reference for identification of particles by LM.08 0. Characteristics of Some Common Light Microscope Objectives Magnification 3 10 50 100 Numerical Aperture 0. Particles that have an index of refraction different from the substrate-mounting material are most easily viewed. It is relatively easy to use as an imaging tool for many applications. values may be 1.25 to 0. surface texture.25 0. Although particles can be resolved and thus observed at the 0. 1973). b Approximate value in green light. For example. Related to particle size.25 "Approximate value with xlO oculars.85 1. 1978). 1980).5 to 2 times larger. Normally.4 Diameter 0 of Field (mm) 9 2 0. Wide field is now common. size and shape can be determined. and birefringence) to help identify a given particle and thus possibly its source (Grasserbauer. the identity of an unknown particle can often be determined. The Particle Atlas (McCrone and Delly. Using the physical properties of particle shape and size and the optical properties of color. 1973) can be used to compare the image of a sample particle to reference micrographs. refractive indices. The shape and size are useful for identifying particle origin. Two additional references containing information on analysis of particles by LM are Steel (1980) and Friedrichs (1986).5 um. fly ash normally appears under a light microscope as spheres. It is the contrast between the background support and the particle that is most often important when trying to view an object by LM.4 0.5 0. color. shape and size determination is most reliably done on particles larger than several micrometers in diameter (Steel. Determination of shape and size often represents the first step in single-particle analysis. crystallographic properties.3 0. Fibers are an exception to the above statement because even with a lateral dimension as small as 0. shape. fractal-like (complex branched-chain) structures are usually formed from a combustion source. optical characterization requires particles to be greater than 5 to 10 um in lateral dimension (Steel. 1980).4 Resolving* Power (M-m) 5 1. and birefringence. and dark. The contrast can be . Size and Shape Analysis. Capabilities and Applications LM is often the first microanalytical technique used to examine a sample because it is a nondestructive approach. A good working rule of thumb is that the maximum magnification of a light microscope is 1000 times the numerical aperture of the objective (McCrone and Delly. Identification by Light Microscopy. refractive index. Sometimes the shape will provide information about the particle type and thus the most probable formation mechanism of a particle. but identifying a material through its optical properties can be difficult. A general detailed reference for LM is given by Chamot and Mason (1958).
Two large objects in the central part of the micrograph are barely visible under these conditions. help to identify birefringent materials in the sample. The advantage of uncrossed polarizers is that the particles are. 12-3. shown in Figure 12-3c. Figure 12-3a illustrates the problem with viewing the filter in direct (unaided) transmission. but in this case the objects take on a three-dimensional appearance. Crossed polarizers. The micrographs are taken in transmitted light. 1986). in the last case (d). When phase contrast microscopy is applied (Fig. Finally.a b c d Fig. c. NIST. 12-3b). the phase shift of the light transmitted through the particles is used to enhance the contrast. Straight transmitted light. Another way to increase the visibility of the particle is by differential interference contrast. the particles become quite visible. b. differential interference. (Courtesy of E.) improved by a number of techniques. Figure 12-3 shows the same field of view of a collected airborne particle sample using different contrast enhancers. and the sample is prepared by treating with acetone vapor to make the filter transparent (Baron and Pickford. on the other hand. would make all of the isotropic materials (such as glass . Set of four light micrographs illustrating various LM techniques that help to increase particle visibility by increasing contrast and. d. still visible in the field of view and the particles made of anisotropic material stand out as illuminated objects. a. for the most part. This is considered a complementary technique to phase contrast. effect of slightly uncrossed polarizers. transmitted light using phase contrast. The filter material is mixed esters of cellulose. Figure 12-3d shows the effect of slightly uncrossed polarizers. Steel. The last frame (d) brings out the birefringent material present in the sample as the apparent luminous objects.
Because the analysis assumes a uniform distribution on the filter.25 |im. 1977). for specific fiber types. For instance. and established quality control procedures are all-important components of proper laboratory practice. Thus. 1952. Although the morphology observed under PCM allows some discrimination between fiber types. these biases may appear as increased variability in the overall results. and the following contributions relating to fibers have been extracted from his work. Detailed discussion of these techniques is not possible here. taking a small portion for microscopic analysis can therefore result in significant variations in the reported concentration. It cannot be used to detect fibers thinner than about 0. Often the observation of fluorescence. but is given in McCrone and Delly (1973). Refractive index is another parameter that can be determined and used as a powerful tool for particle identification. This is the only way that results from one laboratory can be compared reliably with those of other laboratories. in a particle. no background dust interference. or other sampling influences. In fact. most analytical methods in the NIOSH Manual of Analytical Methods state an overall uncertainty (combined variability and bias) of better than 10% (NIOSH.10 (or 10%) is expected. such as polarized light microscopy (PLM) and scanning (SEM) and transmission (TEM) electron microscopy. it is not specific enough to allow positive identification of asbestos or other fibers. 1940). one microscopist may introduce biases relative to other microscopists due to decisions about which particles to count as fibers. It can be used with other analytical techniques. Some of these are due to the small sample size observed as part of the measurement procedure. The use of established analytical procedures for fiber count analysis is extremely important. PCM is primarily used for fiber counting to provide an index of asbestos fiber exposure in workplaces where asbestos is known to be present. Analyses of these kinds require considerable experience to be useful for identifying particle composition. 1984). Several additional techniques are useful in conjunction with LM. Some microscopes are equipped with monochromatic or near-monochromatic light sources that can be used to excite fluorescence. Microscopist training." Microchemical reactions can be used to help identify the particle composition (Seeley. other sources of variability and bias can occur. For instance. proper equipment. the accuracy (including bias and variability) can be no better than this level. The accuracy of various fiber-counting techniques is poor when compared with other analytical methods. When comparisons are made between groups of microscopists. Light that is transmitted through the object is phase shifted relative to that transmitted through the substrate only. usually excited in the ultraviolet. Under optimum analysis conditions (uniform sample deposit. The PCM transforms the phase difference to an intensity difference by forming an interference pattern using the phase shifted and unshifted light. mineral wool and fibrous glass (NIOSH. In addition.or amorphous plastics) "invisible" and show only those particles (as luminous objects) that rotate the polarized transmitted light. Baron (1993) presents an extensive discussion of microscopic techniques for fibers. with accuracy to one part in one thousand (Grasserbauer. To ensure . electrostatic. Fiber Analysis. optimum loading) a relative standard deviation of 0. The refractive index measurement. but intensity differences are detected. if present. fibers may be nonuniformly distributed in the sample due to inertial. PCM also is used for measurement of man-made mineral fibers (MMMF). Measurement Accuracy of Fibers by PCM. The phase shift is not detectable by the eye. provides information for identifying the particle's composition. for example. Chamot and Mason. is accomplished by immersing the particle in a series of index matching fluids to find the matching refractive index that causes the particle to "disappear. 1978). Phase contrast microscopy (PCM) is an interferometic technique that enables particles with low contrast (transparent particles) to be viewed.
1986. and oil-fired soots. To overcome this size limitation. and (4) miscellaneous particles. The number of particles is normally determined per . 1984). Particles remain on the filter. the types of particles are broken down into four categories: (1) wind-blown particles such as fibers and minerals. 1986). 1989. An aliquot of the particles should be tested with the oil to ensure that no reaction or dissolution takes place. some filters can be made transparent or removed entirely to allow viewing with transmitted light. Several formal programs of sample exchange have been established for PCM. Some "filter clearing" agents are given in Table 12-3 (Friedrichs. the particles are mounted on a glass or quartz slide. polycarbonate filters dissolve in chloroform). are important for establishing analytical confidence limits.g. interlaboratory exchange of samples may not be necessary. 1991). either transmitted or reflected light can be used. that is. 1981. In this reference. (3) combustion particles such as auto. as well as performance of blind repeat analyses.S. filters can be treated with index matching fluid. Occupational Safety and Health Administration (OSHA) asbestos fiber exposure level regulations. and the International Asbestos Fibre Regular Interchange Counting Arrangement (AFRICA) program (Institute of Occupational Medicine. both within-laboratory and interlaboratory sample exchanges are necessary (Abell et al. and particles with a refractive index matching the immersion oil will be difficult to see. However. This reference contains over 600 color photomicrographs of particles from various sources and of known composition (McCrone and Delly. the U. 1973).. regular sample exchanges with other laboratories are required. 1986). The usual technique for determining analytical biases. if a study is intended only to provide relative fiber concentrations to show differences with time or location. Each has its own advantage and disadvantage. In a light microscope. the filter is ashed. Thus. leaving the refractory particles behind.'s Regular Inter-Laboratory Counting Exchange (RICE) program (Crawford and Cowie. For a laboratory performing PCM analyses to establish compliance with U.uniformity of application of these analytical procedures. Edinburgh).. the particles no longer have a filter support and must be remounted on a transparent substrate. but the particles usually must be >1 urn.. The authors provide a step-by-step characterization procedure for classifying the particles into one of the four categories. In the last two approaches. Opaque particles and particles with large indices of refraction are most easily viewed in this manner. LeGuen and Galvin.K.. These include the American Industrial Hygiene Association's (AIHA) Proficiency Analytical Testing (PAT) program (Groff et al. The same reference contains scanning electron micrographs of the same 600 particles shown in the photomicrographs. The oil will contaminate the particles prepared in this manner. The number of particles collected on a substrate can be determined and related to the particle concentration in an aerosol. does not work because an alternate fiber-counting technique that measures the "true" fiber concentration does not exist. coal-fired. Baron and Pickford. Measurement of fiber concentrations for comparative use within a single study may not need all the components of a complete quality assurance scheme. the final test of fiber counting accuracy is the comparison of one's results with those of a group of competent laboratories. For transmitted light. Ogden et al. A second method is to dissolve the filter using an appropriate solvent (e. and cleaners. In the third method. Reflected light can be used to view particles collected on an aerosol filter surface. the use of published counting and sample preparation procedures. fertilizers. (2) industrial particles such as abrasives. In the first method. Friedrichs (1986) mentions three ways to transform the filter. Sample Preparation and Practical Applications. The Particle Atlas can be consulted to classify a particle on the basis of its physical and optical properties. the comparison with a reference method. For instance. but liquid particles or particles soluble in the fluid may be dissolved or possibly removed. polymers. so subsequent microanalysis on the particles is unlikely using other techniques. An index or immersion oil may be applied to the sample to improve viewing under transmitted light.
. Carter et al. The particles in Figure 12-4 were collected on a filter consisting of mixed esters of cellulose. This estimate can be related to the airborne particle number concentration (based on sample air volume) as in the case for asbestos number concentration (Asbestos International Association. Capabilities Electron Imaging.625 Polycarbonate Sources: Friedrichs (1986). One of the imaging methods is used in . when a number of randomly selected viewing areas are taken together. and Baron and Pickford (1986). Depth of field usually encountered in LM is not as large as that found in the SEM.44-1. Electron imaging that provides the analyst with particle size and morphology is accomplished by two different methods. In these micrographs.48 1. especially for particle counting. the lower right regions shown in Figure 12-4b become clear images. Note that only some of the asbestos fibers are in focus in the light micrograph while the entire electron-generated image is in focus. and. while others are difficult to see. As the microscope focus is altered. The filter is slightly bowed in the center as a result of air flow through the filter cassette. This bowing causes a distorted planar surface for the microscopy. The relevance of depth of field.584 or 1. is illustrated by the set of micrographs in Figure 12-4.48 1. In Figure 12-4a. ELECTRON BEAM ANALYSIS OF PARTICLES Principle of Electron Beam Excitation A schematic of a typical electron beam instrument and some of its analytical functions is shown in Figure 12-6. both with approximately the same magnification. 1989). certain particles are visible and in focus. Membrane Filter Clearing Agents for Light Microscopy Filter Type Mixed esters of cellulose Clearing Agent Acetone vapor/triacetin (AIA method) Dimethyl formamide/Euparal method Acetone vapor/Euparal method Immersion oils Chloroform-dissolved materials Refractive Index 1. but the lower right is out of focus. The top is a light micrograph and the bottom is an electron micrograph. a series of images can be obtained as "optical sections" of a three-dimensional object. The electrons emitted from the filament are formed into a beam and focused by an ion lens system onto the specimen. By changing the objective to specimen distance.Next Page TABLE 12-3. Le Guen and Galvin (1981). Figure 12-5 contains two micrographs of the same field of view of amosite asbestos. The recent development of the confocal microscope provides a different approach by limiting the image formation strictly to those photons scattered within the depth of field. 1979. The source of the electron beam is an emitter such as a heated tungsten filament shaped into a fine tip. and particles in the upper left gradually become fuzzy and indistinguishable. resulting in the scattering of the beam electrons and the ejection of both electrons and X-ray photons from the specimen.48 1. The electron beam interacts with the atoms of the sample. Clearly a shallow depth of field is problematic. the upper left of the field of view is in focus. Figure 12-4 is an example of a typical LM application that might be employed by an aerosol scientist for examining a filter surface in reflectance. an estimate can be made of the number of particles on the entire filter surface. leading to poor particle detection due to limited depth of focus. viewing area.