Imaging Analysis: Human WNT Ligands in Epiblast and Ectodermal Differentiation

MATLAB code for the quantitative analysis of fixed imaging data described in:

The Role of Human WNT Ligands in Epiblast and Ectodermal Differentiation Eleni Anastasia Rizou and Aryeh Warmflash

Imaging experiments were conducted on an Olympus FV3000 laser scanning confocal microscope to study protein expression and localization in immunostained cell samples. The code provides a reproducible pipeline for analyzing nuclear and membrane protein expression, including nuclear segmentation, intensity profiling, and directional sampling for fixed cells.

Prerequisites

• MATLAB (developed in R2022a)
• Bio-Formats toolbox
• Ilastik — used in the juxtaposition workflow to segment images and generate binary mask files (.h5) for nuclei

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Uniform Experiment — Fixed Cells

Processing pipeline for uniform fixed-cell images.

Pipeline Scripts (UniformAnalysis/)

Run these in order:

1. maxprojectionmaking.m — Generates z-max projection TIFF images.
2. panelmaking.m — Creates individual condition-specific PNG images.
3. A_uni_makingplots.m — Produces expression plots per condition.

Functions (functions/)

Function	Description
reamaxprojectionfun.m	Batch processing for z-max projections from multichannel images (specify 0).
reacompareMultiChannelImagesTiff.m	Generates condition-specific PNG images.
ai_reaExprTablemakingfun.m	Creates expression tables per cell and condition.
aii_reaUnifonexpAllplots.m	Generates bar plots from expression tables.
aii_MutltreaUnifonexpplots.m	Creates bar plots from multiple experiment replicates.
reaConcenLineplots.m	Produces line plots showing expression variation across time points or concentrations.

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Juxtaposition Experiment — Fixed Cells

Scripts for analyzing contact (juxtaposition) experiments from scratch.

Pipeline Scripts (BorderAnalysis/3DcontactProtocol/)

Run these in order:

1. A_contact_stitch3Dmaking.m — Generates z-max projection TIFF images.
2. B_contact_maxprojectionmaking.m — Creates individual condition-specific PNG images.
3. C_contact_panelmaking.m — Produces expression plots per condition.
4. Ilastik training (external software) — Train on all channels to generate nuclear masks (.h5 files). Save the training output in:
   • IlastikTraining2 for the sender-based segmentation approach.
   • IlastikTraining for single-cell segmentation.
5. D_contact_bordermaking.m — Uses the Ilastik-generated masks in subsequent analyses.

Functions (functions/)

Function	Description
reastitchfun3DMXN	Performs 3D stitching of image tiles.
reamaxprojectionfun	Batch processes z-max projections from multichannel images.
reacompareMultiChannelImagesTiff.m	Generates condition-specific PNG images.
readSepChanTifffun	Separates each channel to facilitate Ilastik training in cell segmentation.
d_reaContDistTabmakefun.m	Calculates object intensities, generates line plots per condition/gene, and creates summary tables for further analysis.
d_reaMergeContacTabfun.m	Normalizes experimental results to prepare data for statistical analysis.