The microscope uses lenses to magnify and focus the image of objects that are beyond the resolution of the human eye. These lenses may be optical, as in the case of the light microscope, or electromagnetic, as in the case of the electron microscope. The practical limit to the resolution of the image of a particular object is dependent on the wavelength of light or the energy of emitted electrons. Fig. 12 shows schematic diagrams of the complex optical microscope and the scanning electron microscope .
Filtration is a suitable method for the collection of aerosols for microscopy, as mentioned previously. Particles may also be collected using inertial devices and may be examined by microscopy . A third method that has not been mentioned is electrostatic precipitation, or sampling.
Electrostatic precipitators collect particles that may subsequently be examined by microscopy. The method precipitates particles according to the charge they carry. The aerosol is passed between two plates or surfaces across which is a large potential difference. Deposition occurs as a function of the ratio of the charge to the inertia (mass and velocity) of the particles . The principle on which this method is based considers the motion of particles in planes directly perpendicular to and parallel to the plane of the condenser .
The charge on a particle may originate in various ways. In crushing powders, the particles receive electric charges, as might occur in jet milling; the atomization of liquids produces droplets that are charged owing to fluctuations in the concentration of ions in the liquid, such as occurs in nebulization; aerosols formed at high temperature are charged by thermoionic emission; and precipitation of gaseous ions and electrons or aerosol particles produces charges. At the moment when aerosols are formed, particles may be highly charged, but, whatever the initial distribution of charges, a stationary state is gradually approached due to precipitation of the particles or ions that are constantly being formed.
The TSI Model 3100 Electrostatic Aerosol Sampler collects and deposits particles ranging from 0.02 to 10 mm onto a collection substrate. It does not collect large samples quickly. The samples may be collected directly onto glass slides, metal foil, plastic sheets, or coated electron microscope grids.
This has the advantage of eliminating the hazard of contaminating the samples during transport. Fig. 13 shows a diagram of the arrangement of the instrument. The aerosol is drawn into the chamber of the instrument at a rate of 5 L/min, which must be precalibrated by the operator. Positive ions are generated by corona discharge from a fine, positively charged tungsten wire. As aerosol passes through the charging section, intermittent pulses of these ions impart a positive charge to the particles. The intermittent pulses are produced by an alternating voltage. During the negative phase, positive ions mingle with aerosol particles that, in turn, become positively charged. As the aerosol leaves the charging section, it enters the collecting section. If no voltage difference exists between the top and bottom of the collecting section, the aerosol passes through and does not deposit. In the instrument's intermittent, precipitation mode, particles are located on the substrate in an unbiased (by morphological characteristics, including particle size) manner during collection. The sampler applies a positive square-wave voltage to the upper polished charging plate. The positive square wave is "on" for 1.5 sec and "off' for 3 sec per cycle. The lower plate remains at ground potential. When the square wave is "off," the particles pass through the collection section in the airflow. When the square wave is "on," the particles migrate from the upper to the lower plate. While the positive precipitating voltage is "on" (not in the intermittent mode), particles that are entering the collection section deposit, predominantly according to size and shape. There is an area on the
lower plate that is unbiased by these particle characteristics, and it is marked for the convenience of the investigator. The preferential deposition at the entrance does not exhibit the sensitivity required for this to be useful as a particle-sizing technique per se. Particle deposition in the entrance region is concentrated enough to allow collection for chemical or radioactivity analysis. Particle sizing must be performed using an independent technique, such as optical or electron microscopy .
Traditionally, particles were sized by "looking at them." Initially, as indicated by early pharmaceutical requirements [90,91], the compound, transmission light, microscope, and its various corollaries were used. These include bright- and dark-field, polarized, reflectance (incident), differential-interference, and phase-contrast illuminations. These techniques pose some immediate questions of data interpretation. Heywood [92,93] was one of the first investigators to suggest that to avoid the complexities of shape, a standard should be adopted that normalized the image of the particle to circular disks of equivalent area. Specialized graticules were developed in which circular disks were placed in the field of view of the microscope for direct comparison with the particle being examined [9,19,94]. Fig. 14 shows one example of the numerous similar graticules that were developed.
There are many limitations to this technique. It is a subjective measure that is open to observer errors. The time taken to measure individual particles in this way restricts the total number of size estimates that can be collected, allowing statistical errors to occur . Indeed, the American Society for Testing and Materials (ASTM) recommends that the modal class of the size distribution should contain at least 100 particles and that at least 10 particles should be present in each size class . Another approach, that of "stratified sampling," recommends at least 10 particles in every size class that has a significant influence on the size curve . Microscopy, as a static method, can identify irregular shapes but cannot fully predict their effects on particle behavior. Similarly, it does not take into account density, which plays a role in the dynamic behavior of particles. Both of these factors influence aerosols entering the lung . There are practical limits to the accuracy of measurements. The resolution of the optical microscopes make them suitable for measurement of particles 1 mm or larger in size. Finally, microscopy examines particles in their plane of maximum stability, and this two-dimensional view may not accurately reflect the three dimensions of the particle.
Image-splitting optical microscopy was developed to reduce observer error [Watson image-shearing eyepiece , Fleming particle size analyzer ]. These
methods use mirrors or prisms that may be combined with a video camera to create two images of the particles being viewed. One image may be displaced with respect to the other, which remains stationary. By displacing the particle image so that its opposite sides touch (i.e., moving the right edge to touch the left edge of the stationary particle, or vice versa), the distance the image is moved represents a measure of the particle size. This method is still limited by the resolution of the microscope, although the subjectivity of the estimate of the particle size is much reduced.
The wavelength of the visible spectrum used in optical microscopy, 400700 nm , is much larger than that of an electron of Compton wavelength, 0.386 pm . Materials approaching in size the wavelength of the energy form used will have a reduced tendency to absorb or interfere with its path. Ultimately, a limit to detection, where no absorbance or interference occurs, will be reached in which the material will become essentially "invisible." It is apparent that this will happen at much larger sizes for visible light, photons, than for electrons. Indeed, there is an approximately 1000-fold difference between the two techniques. Thus, electron microscopy allows greater resolution and, in turn, magnification than optical microscopy.
Scanning electron microscopy is open to some error due to the gold-, palladium-, or platinum-coating methods used . The coating not only adds slightly to size of the particles but may mask morphological features that could influence particle behavior. Transmission electron microscopy does not exhibit this drawback. Figure 15 shows scanning and transmission electron micrographs of samples from the same population of particles. The particles appear relatively smooth by scanning electron microscopy and rough by transmission electron microscopy. The smoothness in this instance may be an artifact of gold coating, which in this case may have allowed greater definition of surface morphology. However, this example is given to indicate the care in preparation and the caution that should be exercised in interpreting electron micrographs.
The major advantage of microscopic methods is their direct measurement of particle size. In many of the alternative methods, at least one automated datainterpretation or calculation step is inserted between the instrumental analysis and establishing the estimate of particle size. This reduces the subjectivity of the measurement while increasing the likelihood of interpretive errors.
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