Differential Counting of Asbestos Using Phase Contrast and Fluorescence Microscopy

Considering the increasing use of various asbestos substitutes, asbestos risk management in many industries may require accurate techniques for detecting and distinguishing asbestos from non-asbestos fibers. Using fluorescently labeled asbestos-binding proteins, we previously developed a novel method for detection and counting of asbestos fibers under fluorescence microscopy (FM). This method can provide speedy, on-site detection and identification of the asbestos fibers and has higher sensitivity than phase contrast microscopy (PCM). However, current asbestos exposure limits are derived from risk assessments based on epidemiological studies that were conducted using PCM fiber counts. Therefore, the sensitivity of asbestos testing should be maintained at PCM level to properly assess compliance with these limit values. Here, we developed and tested a novel application of FM as a differential counting method that complements PCM analysis and is fully compatible with the PCM-based epidemiological data. In the combined PCM-FM method, the fluorescent asbestos-binding probe is applied prior to filter clearing. The method makes it possible to easily switch between two microscopic techniques while analyzing the same fields of view: PCM is used for counting fibers, and FM for differentiating asbestos from non-asbestos fibers. Using airborne dust samples from demolition sites in Japan, we compared PCM-FM with scanning electron microscopy (SEM)-based differential counting method. Statistical analysis indicated a slight conservative bias of PCM-FM method, combined with relatively high variability across the full range of fiber concentrations in our sample set. Using correlative microscopy, we also evaluated the specificity of FM staining, which is a potential cause of variability between the two methods. The energy-dispersive X-ray analysis indicated that ~95% of fluorescently stained fibers in the demolition site samples were correctly identified as asbestos. While further research is needed to fully clarify the causes of variability between FM- and SEM-based differential counting, PCM-FM could be used for rapid and selective detection of asbestos fibers in field samples.