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Air pollution, especially particulate matter (PM), is a significant environmental pollution worldwide. Studying the chemical, environmental, and life-related cellular physical characteristics of size-fractionated PMs is important because of their different degrees of harmful effects on human respiratory tracts and organ systems, causing severe diseases. This study evaluates the chemical components of size-fractionated PMs down to PM 0.1 collected during a biomass-burning episode, including elemental/organic carbon and trace elements. Single particle sizes and distributions of PM 0.1 , PM 0.5-0.1 , PM 1.0-0.5 , and PM 2.5-1.0 were analyzed by scanning electron microscopy and Zeta sizer. Two commonly used cell lines, e.g., HeLa and Cos7 cells, and two respiratory-related cell lines including lung cancer/normal cells were utilized for cell cytotoxicity experiments, revealing the key effects of particle sizes and concentrations. A high-speed scanning ion conductance microscope explored particle-stimulated subcellular physical characteristics for all cell lines in dynamics, including surface roughness (SR) and elastic modulus ( E ). The statistical results of SR showed distinct features among different particle sizes and cell types while a E reduction was universally found. This work provides a comprehensive understanding of the chemical, environmental, and cellular physical characteristics of size-fractionated PMs and sheds light on the necessity of controlling small-sized PM exposures.