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[Display omitted] • Visualization of microplastics in crayfish using LCM-Raman imaging. • Microplastics were detected in the gills, intestines, and ovaries of crayfish. • Crayfish exposed to 9 mg/L of microplastics accumulated 13,000 particles in 28 days. • RPV values can quantitatively evaluate the contamination level of microplastics. Crayfish is one of the most important freshwater aquaculture species in China. The potential risks of crayfish consumption caused by environmental microplastic pollution have attracted much attention. In this study, a total of 72 crayfish samples were exposed to the microplastic concentrations of 1 mg/L, 3 mg/L, and 9 mg/L for 7, 14, and 28 days, and microplastic contamination levels in crayfish were then explored by laser confocal micro-Raman (LCM-Raman) imaging and scanning electron microscope (SEM). LCM-Raman imaging showed better performance in microplastics identification. Besides, the average percentage of the contaminated area in visualized LCM-Raman images was used to quantitatively assess contamination levels. Following 28 days of exposure to 9 mg/L microplastics, microplastic accumulation reached about 13,000 particles per crayfish. The results confirmed that LCM-Raman imaging combined with image processing technology could be used to construct a high-performance analytical strategy for the assessment of microplastic contamination in crayfish. [ABSTRACT FROM AUTHOR]