Preprocessing

Overview

Preprocessing is an essential step to prepare your microscopy data for analysis in Celldetective. It includes both off-software and in-software methods to align, correct, and optimize your stacks for segmentation and downstream measurements.

Registration

Stage drift and shaking make still objects move across the frames, which misleads tracking and blurs time-averaged measurements. We highly recommend registering the movies before segmentation. Celldetective registers stacks by phase cross-correlation, directly in the Preprocessing module (see the table below). The stacks can also be aligned before being imported, with external tools like Fiji (ImageJ), which can correct rotations too.

In-software preprocessing

The Preprocessing module lets you batch-correct stacks directly within Celldetective. Corrected stacks are saved with the prefix Corrected_. The corrections added to the list are applied in order: the first one reads the raw movie, the next ones correct the Corrected_ stack in place. Each step is recorded with its parameters in the log_preprocessing.txt file of the position.

Task	Description	Guide
Background correction (Model Fit)	Fits a 2D surface to the background, excluding cells. Best for fluorescence images.	how-to
Background correction (Model Free)	Computes a median background from multiple positions or timeframes. Best for brightfield/reflection microscopy.	how-to
Channel offset correction	Aligns channels that have pixel shifts between modalities.	how-to
Stack registration	Corrects the drift of the field over time, estimated on one channel and applied to all.	how-to

For a full list of parameters (thresholds, tile options, etc.), see the Preprocessing Protocols Reference.

Bibliography

Schindelin, J., Arganda-Carreras, I., Frise, E. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676–682 (2012). https://doi.org/10.1038/nmeth.2019