Chapter 4 88 Standardize the range of detected uEV size After knowing the size range and marker of uEV in the unprocessed urine samples, a step-by-step gating strategy was developed to distinguish single CD63+ uEV particles from non-uEV components or coincidence events in IFCM. As demonstrated with NTA (Figure 2B), we chose the detection range of <1200 nm to include virtually all uEVs. Based on the previously published calibration of our IFCM, an SSC cutoff value of 5279.179 arbitrary units, corresponding to 1200nm (diameter) EVs, was obtained and used to include uEVs ≤ 1200 nm for all the following analyses.23 Figure 2 - uEV characteristics tested by TEM, NTA, and TR-FIA in the HC urine (n = 5 ). A) uEV-like structures in TEM. B) The size distribution of urinary particles in NTA. C) uEV tetraspanin levels as reported by TR-FIA. *p < 0.05. Exclude multiplets and false singlets The fluorescence intensity of uEVs in multiplets cannot be individually characterized.28 Following previous research, we also initially tried to utilize intensity masks to calculate the spot count feature.22,23 However, the Intensity Mask mistook close doublets as singlets (Supplementary Figure S3A). Compared with Intensity masking, Peak Mask only selects pixels with peak intensity (Supplementary Figure S3B).28 Therefore, spot numbers could be more precisely quantitated using the Peak Mask. The spot-to-background ratio of the Peak Mask should not be higher than the value of 1, lest the number of individual spots are underestimated. When the ratio was 3, this Peak mask ignored pixels with < 3-fold of the average intensity of the whole image, leading to missing dim spots (Supplementary Figure S3C). Notably,“<” of this ratio has no meaning because in the Peak mask
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