Free orbicules of Cupressaceae detected in daily aerobiological samples by optical and confocal microscopy

Members of Cupressaceae and Taxaceae are known to release large amounts of highly allergenic pollen grains into the atmosphere, which are responsible for the onset of pollinosis in many countries throughout the world. In addition to pollen grains, their pollen sacs produce orbicules, which are submicron particles reported to carry allergens and which are potentially able to reach much further down the respiratory tract than pollen grains. Previous research has postulated the presence of orbicules in the atmosphere; however, direct observations have not yet been reported. The aim of this research was to provide the first direct evidence that Cupressaceae orbicules are released into the atmosphere by detecting them in daily aerobiological samples. We observed pollen sacs, pollen grains, and orbicules of nine species of Cupressaceae using scanning electron microscope (SEM). We then used a light and confocal microscope, to examine daily aerobiological samples. Under SEM, we measured the orbicule size (0.494â0.777 µm) and detected unknown nanometric particles (130â200 nm). Under the light microscope, aerobiological samples showed clusters of stained dots surrounding the pollen grains of Cupressaceae. Under the confocal microscope, the same clusters were resolved into submicron particles with the same autofluorescence as the pollen grains. These features enabled us to identify them as orbicules. We believe that our findings help to explain the onset of pollinosis and allergic asthma related to Cupressaceae pollen grains in many countries, even before pollen grains are actually detected or after they are no longer observed in aerobiological monitoring samples