PB1399 - Automated segmentation and quantitative analysis of Weibel-Palade body morphology, localization and content using CellProfiler

Background: One of the most used and versatile methods to study number, dimensions, content and localization of secretory organelles is confocal microscopy analysis. However, considerable heterogeneity exists in the number, size and shape of secretory organelles that can be present in the cell, as is the case for Weibel-Palade bodies (WPBs), the cigar-shaped secretory organelles of endothelial cells. One thus needs to analyze large numbers of organelles for valid quantification of these parameters.

Aims: For this purpose we aimed to develop an automated, unbiased method to process and quantitatively analyze organelle morphology and content from microscopy data.

Methods: The open source software CellProfiler was used to establish two analysis pipelines, called OrganelleProfiler and OrganelleContentProfiler, aimed at quantification morphometric parameters and content of organelles, respectively. Endothelial colony forming cells (ECFCs) stained for WPB content protein Von Willebrand factor (VWF), cell junctional marker VE-cadherin, endoplasmic reticulum marker PDI and WPB maturation marker Rab27A were imaged by confocal microscopy and analyzed using OrganelleProfiler and OrganelleContentProfiler pipelines. Cell profiler measurements were checked for validity by manual quantification using Fiji image analysis software.

Results: We used OrganelleProfiler to quantify cell count and size, and WPB count, size, shape, and relative position to cell nucleus and periphery on a cell-to-cell basis (figure 1). Characterization of two distinct phenotypic classes of ECFCs using OrganelleProfiler showed significant differences in WPB morphometry and localization (figure 2). Using OrganelleContentProfiler to quantify secondary signals located in or on the organelle or in the cytoplasm we were able to establish that Rab27A but not PDI are found in or on WPBs.

Conclusion(s): To conclude, the OrganelleProfiler and OrganelleContentProfiler pipelines provide powerful high-processing quantitative tools for analysis of cell and organelle characteristics.

Funding: Netherlands Organization for Scientific Research (NWO) in the framework of the NWA-ORC (NWA.1160.18.038) and from the Landsteiner institution for blood transfusion Research (LSBR-1707).