A 0.01% limit of detection can be observed in CLARITY processed cell pellets. (a) The mixed cell pellets ratios generated with HEK293-GFP and SUP-T1 cells. (b) A representative mixed cell pellet imaged before clearing and 10 days after clearing under a dissecting microscope. (c) Confocal microscopy (Leica SP8) images of the cell pellets at different ratios (25X). Top row: 3D z-stack images, Bottom row: a corresponding representative 2D optical section image. The HEK293-GFP cells (green) can be detected in the FOV for all ratio groups, including the rare event level of 0.01%. (d) Manual counting of HEK293-GFP (green) was accomplished by calculating the ratio of positive GFP cells/total DAPI (blue) cells (optical sections were used for manual counting, n ≥ 5 for each sample). Statistics were calculated by Microsoft Excel and GraphPad Prism 7.0. (e) The automated analysis and quantification of the GFP positive cell pellets was performed using a spot detection strategy generated with Imaris 9.1.2 software.