Alignment of multiple 3D z-stacks to build a

Our team is planning a Confocal Imaging experiment and we would utilize Cell Profiler for post-acquisition analysis.

The imaging experiment we want to design is as follows,

• We have cells growing in a soft gel-like substrate or scaffolds such as agarose.
• Sectioning will be done from 5microns to 15microns, which will give us tissue like samples
• The sections/tissue/cells will be either fluorescently labeled or unstained
• We will then perform confocal z-stack imaging on individual sections. For instance, section1 ,2 ,3 … (different sections from 1 thick sample)
• We want to align all z-stacks of different sections to reconstruct a 3D model for a single sample. Z-stack section1 + Zstack section 2……

Analysis goals

Kindly assist me if doing so is possible, we don’t want to superimpose them or get an average image, rather we want to align them together and construct a 3D model. Please also let me know if we can use different strategies in experimental designing or analysis modalities.


I have already tried zerene stacker, imageJ, Confocal NIS-elements, but they are giving an average of all images or superimposing them. What I want is to build 3D models from multiple z-stacks and align them together in a sequence.

HI @Hadiqa,

Sounds like an interesting challenge!

You can use CellProfiler to analyze 3D Z stack images but our software is focused on segmentation and measurement of individual images. To my knowledge, we don’t have a module or plugin for aligning 3D images.

If you’re interested in getting started with CellProfiler, I’d recommend this introductory workshop and 3D tutorial available on the Center for Open Bioimage Analysis Youtube channel: I2K 2020 tutorial: Introduction to Image Analysis and Machine Learning With CellProfiler and Cell... - YouTube

Good luck,

If by alignment your stacks just need to translated (aka not warped), there’s a way you COULD do it in CellProfiler but it’s a little messy. Do they just need to be moved in X-Y, or also warped?

Have you tried StackReg or MultiStackReg in FIJI? Those are my favorite plugins for registration- I’ve used them for registering time points but a priori there’s no reason to think they won’t work just as well at aligning consecutive Z planes.