Seattle Asbestos Test, LLC performs asbestos testing for bulk material using Polarized Light Microscopy (PLM) utilizing various testing methods, and asbestos fiber counting in air samples using Phase Contrast Microscopy (PCM). In addition, Seattle Asbestos Test also performs Rotameter Calibration, and Respirator fit test. Seattle Asbestos Test is also developing programs for Lead and Metals Testing, and Mold and Particulates Identification.
For Polarized Light Microscopy (PLM), the tests available at Seattle Asbestos Test include the following:
PLM by EPA 600/R-93/116
PLM by EPA 600/M4-82-020
PLM by NIOSH 9002
PLM by OSHA ID191
EPA Point Count 400 and 1000
EPA Gravimetric Point Count
For Phase Contrast Microscopy (PCM), Seattle Asbestos Test mainly performs PCM by NIOSH 7400.
As requested by more and more clients, Seattle Asbestos Test started Rotameter Calibration and Respirator Fit Test services.
Lead and Metals Testing and Mold and Particulates ID are currently under development, and they should be available in the near future.
Introduction to Polarized Light Microscopy (PLM)
The polarized light microscope is designed to observe and photograph specimens that are visible primarily due to their optically anisotropic character. In order to accomplish this task, the microscope must be equipped with both a polarizer, positioned in the light path somewhere before the specimen, and an analyzer (a second polarizer), placed in the optical pathway between the objective rear aperture and the observation tubes or camera port. Image contrast arises from the interaction of plane-polarized light with a birefringent (or doubly-refracting) specimen to produce two individual wave components that are each polarized in mutually perpendicular planes. The velocities of these components are different and vary with the propagation direction through the specimen. After exiting the specimen, the light components become out of phase, but are recombined with constructive and destructive interference when they pass through the analyzer. Polarized light is a contrast-enhancing technique that improves the quality of the image obtained with birefringent materials when compared to other techniques such as darkfield and brightfield illumination, differential interference contrast, phase contrast, Hoffman modulation contrast, and fluorescence.
Polarized light microscopes have a high degree of sensitivity and can be utilized for both quantitative and qualitative studies targeted at a wide range of anisotropic specimens. Qualitative polarizing microscopy is very popular in practice, with numerous volumes dedicated to the subject. In contrast, the quantitative aspects of polarized light microscopy, which is primarily employed in crystallography, represent a far more difficult subject that is usually restricted to geologists, mineralogists, and chemists. However, steady advances made over the past few years have enabled biologists to study the birefringent character of many anisotropic sub-cellular assemblies.
How to Achieve Polarization of Light - Natural sunlight and most forms of artificial illumination transmit light waves whose electric field vectors vibrate in all perpendicular planes with respect to the direction of propagation. When the electric field vectors are restricted to a single plane by filtration then the light is said to be polarized with respect to the direction of propagation and all waves vibrate in the same plane.
What is Optical Birefringence - Anisotropic crystals have crystallographically distinct axes and interact with light in a manner that is dependent upon the orientation of the crystalline lattice with respect to the incident light. When light enters a non-equivalent axis in a anisotropic crystal, it is refracted into two rays each polarized with the vibration directions oriented at right angles to one another, and traveling at different velocities. This phenomenon is termed double- or bi-refraction (or birefringence) and is seen to a greater or lesser degree in all anisotropic crystals.
How to Configure Polarized Microscope - Although similar to the common brightfield microscope, the polarized light microscope contains additional components that are unique to instruments of this class. These include a polarizer and analyzer, strain-free objectives and condenser, a circular graduated stage capable of 360-degree rotation, Bertrand lens, and an opening in the microscope body or intermediate tube for compensators, such as a full-wave retardation plate, quartz wedge, or quarter-wavelength plate. Removal of the polarizer and analyzer (while other components remain in place) from the light path renders the instrument equal to a typical brightfield microscope with respect to the optical characteristics.
Our Olympus BHT Polarizing Microscope - The Olympus BHP polarizing microscope is equipped with all of the standard recommended accessories for examination of birefringent specimens under polarized light. These include a binocular observation tube mounted on the standard intermediate attachment, which houses the selectable and focusable Bertrand lens, as well as a 180-degree rotatable analyzer, allowing both conoscopic and orthoscopic examination of birefringent specimens. The objectives (typically ranging in magnifications of 4x, 10x, 20x, 40x, and 100x) are of the strain-free low birefringence type optimized for polarizing light microscopy, and are mounted in specialized adapters that allow each to be individually centered. The circular 360-degree rotatable stage can be clamped at any rotation position and is also equipped with a centering function, as well as with an accessory mechanical positioning attachment that enables precise x-y translation of specimens.
How to Align Polarized - In polarized light microscopy, proper alignment of the various optical and mechanical components is a critical step that must be conducted prior to undertaking quantitative analysis between crossed polarizers alone, or in combination with retardation plates and compensators. Several essential components must be correctly positioned with respect to both the microscope optical axis and to other mechanical and optical components. The series of alignment steps outlined in this section are intended for general use with polarized light microscopes and should be applicable to both student and research-level instruments.
What Are Compensators and Retardation Plates - Optical anisotropy is studied in the polarized light microscope with accessory plates that are divided into two primary categories: retardation plates that have a fixed optical path difference and compensators, which have variable optical path lengths. Addition of a retardation plate or compensator to the polarized light microscope produces a highly accurate analytical instrument that can be employed to determine the relative retardation (often symbolized by the Greek letter G) or optical path difference between the orthogonal wavefronts (termed ordinary and extraordinary) that are introduced into the optical system by specimen birefringence. The terms relative retardation, used extensively in polarized light microscopy, and optical path difference (D or OPD), are both formally defined as the relative phase shift between the orthogonal wavefronts, expressed in nanometers.
What Is Michel-Levy Birefringence Chart - The birefringence of a anisotropic material can be estimated when observed and/or photographed in a polarized light microscope. A relationship between interference color and retardation can be graphically illustrated in the classical Michel-Levy interference color chart, presented in this section. The graph plots retardation on the abscissa and specimen thickness on the ordinate. Birefringence is determined by a family of lines that emanate radially from the origin, each with a different measured value of birefringence corresponding to thickness and interference color.
Introduction to Phase Contrast Microscopy (PCM)
Phase contrast is an excellent method for enhancing the contrast of thin, transparent specimens without loss of resolution, and has proven to be a valuable tool in the study of dynamic events in living cells. Prior to the introduction of phase contrast optical systems, cells and other semi-transparent specimens were rendered visible in brightfield microscopy by artificial staining techniques. Although these specimens can be observed and recorded with darkfield and oblique illumination, or by defocusing a brightfield microscope, this methodology has proven unreliable in providing critical information about cellular structure and function.
The technique of phase contrast is widely applied in biological and medical research, especially throughout the fields of cytology and histology. As such, the methodology is utilized to examine living cells, tissues, and microorganisms that are transparent under brightfield illumination. Phase contrast enables internal cellular components, such as the membrane, nuclei, mitochondria, spindles, mitotic apparatus, chromosomes, Golgi apparatus, and cytoplasmic granules from both plant and animal cells and tissues to be readily visualized. In addition, phase contrast microscopy is widely employed in diagnosis of tumor cells and the growth, dynamics, and behavior of a wide variety of living cells in culture. Specialized long-working distance phase contrast optical systems have been developed for inverted microscopes employed for tissue culture investigations. Other areas in the biological arena that benefit from phase contrast observation are hematology, virology, bacteriology, parasitology, paleontology, and marine biology.
Industrial and chemical applications for phase contrast include mineralogy, crystallography, and polymer morphology investigations. Colorless microcrystals, powders, particulate solids, and crystalline polymers, having a refractive index that differs only slightly from that of the surround immersion liquid, are often easily observed using phase contrast microscopy. In fact, quantitative refractometry is often utilized to obtain refractive index values and for identification purposes. Other commercial products scrutinized by phase contrast optical techniques include clays, fats, oils, soaps, paints, pigments, foods, drugs, textiles, and other fibers.
Incident light phase contrast microscopy, although largely supplanted by differential interference contrast techniques, is useful for examination of surfaces, including integrated circuits, crystal dislocations, defects, and lithography. A good example is the stacking faults in silicon epitaxial wafers, which are of tremendous significance to the semiconductor industry. In reflected light phase contrast systems, an image of the illuminating annulus is projected into the rear focal plane of the objective, where the phase plate is normally located. In addition, the phase plate is not positioned within the objective, but an image of the rear focal plane is formed by an auxiliary lens system that avoids reflections and scattering generated by the phase plate. It should be noted that in reflected light phase contrast microscopy, phase differences arise from relief on the specimen surfaces, rather than phase gradients within the specimen.
Reduction in halo and shading-off artifacts remains a primary concern in phase contrast microscopy. Apodized phase plates are useful for reducing the severity of halo, and specialized variable phase contrast systems can be fine-tuned to control these effects in order to optimize image quality and the fidelity of information obtained by the technique. There is also considerable interest in development of advanced phase contrast systems that provide accurate measurements of phase specimens having large optical path differences, as well as combined observations with other contrast-enhancing techniques. In particular, phase contrast is often utilized with fluorescence imaging to determine the locations of fluorophores, and shows promise for enhancing contrast in scanning optical microscopy
Introduction to Point Count
Revisions to the Asbestos NESHAP were promulgated on November 20, 1990 and included a requirement to perform point counting to quantify asbestos in samples where the asbestos content is below ten percent. This requirement has been the subject of many questions, and the attached guidance document has been developed to clarify when point counting is required.
It should be understood that while the point count rule was published as a revision to the Asbestos NESHAP, the intent of the revision is to improve the quantitative analysis of asbestos for all applications. Therefore, the revision is required for all NESHAP monitoring, under the conditions discussed in the attached clarification, and recommended for AHERA and other asbestos monitoring application.
First, a sample in which no asbestos is detected by polarized light microscopy (PLM) does not have to be point counted. However, a minimum of three slide mounts should be prepared and examined in their entirety by PLM to determine if asbestos is present.
Second, if the analyst detects asbestos in the sample and estimates the amount by visual estimation to be less than 10%, the owner or operator of the building may (1) elect to assume the amount to be greater than 1% and treat the material as asbestos-containing material or (2) require verification of the amount by point counting.
Third, if a result obtained by point count is different from a result obtained by visual estimation, the point count result will be used.