An ImageJ/FIJI Preprocessing Workflow for Multi-Series Confocal Microscopy Datasets Prior To CellProfiler Analysis

The overall aim of this workflow is to generate three-slice maximum-intensity projections (MIPs) in an effort to preserve weak, spatially localized signals that can be missed when projecting across the entire Z-stack. For this purpose, a two-step ImageJ/FIJI workflow was developed. In the first step, a multi-series .czi file is treated as a container of independent image datasets, and each embedded dataset is extracted and exported as a standalone OME-TIFF file, preserving all acquired channels as full Z-stacks. In the second step, these per-dataset stacks are processed to generate three-slice maximum-intensity projections for user-defined channels. The resulting two-dimensional images are saved with a consistent naming scheme, enabling straightforward downstream grouping and high-throughput quantitative analysis using CellProfiler