Figure 1

The basic normalization process of Intensify3D for 2-Photon and Light-Sheet 3D imaging (a). Left panel. 2-photon imaging setup illustrating the decay in excitation laser (red) and emitted light (green) through the imaged tissue. Red frame, middle panel. 3D projection of In vivo 2-photon Z-stack of CChIs up to 300 µm depth, bottom portion is deeper. Green frame, right panel. 3D projection of image stack post normalization with Intensify3D; note the enhanced visibility of deep neurites (b). Intensify 3D processing pipeline for 2-Photon and light sheet image stacks. The latter requires an additional step to only account for tissue pixels in the image. The images in the stack are normalized one by one (XY normalization). After all the images are corrected the entire stack is corrected (Z Normalization) by semi-quantile normalization (other options exist) (c). Left panel. Light-Sheet imaging setup where the excitation light is orthogonal to the imaged surface. Red frame, middle panel. iDISCO immunostaining and clearing of CChIs as well as striatal Cholinergic interneurons. Original image suffers from fluorescence decay at increasing tissue depth. Green frame, right panel. Intensify3D Normalized image stack. Images before and after normalization are presented at the same brightness and contrast levels.