HeLa cells stably expressing pEGFP-N1-32x were fixed and permeabilized 8 h or 24 h after microporation with 5 µM 2Me/PSLOOP MBs. smFISH was then performed with a pool of probes designed to target EGFP to assess MB detection accuracy for single RNA transcripts. A. Representative maximum intensity projection images of 2Me/PSLOOP MBs (Atto647NN-labeled) and EGFP smFISH probes (TAMRA-labeled) at 8 h or 24 h after microporation (Scale bar, 10 µm). B. The percentage of MB signals that colocalized with smFISH signals (left) and the percentage of smFISH signals that colocalized with MB signals (right) was analyzed on a cell-by-cell basis using a custom MATLAB program. Data are presented as mean ± SD from at least 10 cells. Significant difference from the 2Me/PSFULL MBs is indicated with asterisks (P < 0.05). The images are reproduced with permission from Elsevier .