Abstract
Despite the continuous and intensive development of laboratory techniques, a light microscope is still the most common tool used in pollen grains differentiation. However, microscopy is time-consuming and needs well-educated and experienced researchers. Other currently used techniques can be categorised as images and non-images analysis, but each has certain limitations. We propose a new approach to differentiate pollen grains using the Imaging Flow Cytometry (IFC) technique. It allows for high-throughput fluorescence data recording, which, in contrast to the standard FC, also enables real-time control of the results thanks to the possibility of digital image recording of cells flowing through the measuring capillary. The developed method allows us to determine the characteristics of the pollen grains population based on the obtained fluorescence data, using various combinations of parameters available in the IDEAS software, which can be analysed on different fluorescence channels. On this basis, we distinguished pollen grains both between and within different genera belonging to the Betulaceae, Oleaceae, Urticaceae and Asteraceae families. Thereby, we prove that the proposed methodology is sufficient for accurate, fast, and cost-effective identification and potentially can be used in the routine analysis of allergenic pollen grains.
Highlights
Aerobiology is an interdisciplinary branch of science covering issues related to the movement of biotic elements and particles of organic origin in the atmosphere, including primarily the study of their source, dispersal, and impact on living organisms and the environment [1,2]
Given the applicability of imaging flow cytometry in aerobiology, we investigated the possibility of distinguishing the allergenic pollen chosen using technique
The collected pollen grains were subjected to routine microscopic examination under a light microscope (Supplementary Figures S1 and S2)
Summary
Aerobiology is an interdisciplinary branch of science covering issues related to the movement of biotic elements and particles of organic origin in the atmosphere, including primarily the study of their source, dispersal, and impact on living organisms and the environment [1,2]. Traditional flow cytometry does not have a well-validated real-time control system of the origin of the fluorescent signal because the results are based solely on the parameters received from the device without the possibility of optical verification of the analysed objects [24] This technique cannot distinguish plant species, but it has been noticed that it gains this ability after combining with microscopy [35,36]. The ability to pass up even to 5000 particles per second through the measuring capillary, coupled with the possibility of analysing up to 10–15 spectral bands and additional scatter signals, can enable quick and efficient analysis of multiple parameters [39,40] This method for cell detection usually requires the use of dyes intercalating with their nucleic acids, it is not necessary for pollen grain analysis because they can generate autofluorescence themselves.
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