Postdoc Tracing Weibel-Palade Bodies in Disease using Multi-beam Volume Electron Microscopy

Postdoc position: Unraveling the life cycle of Weibel-Palade Bodies in disease using multi-beam volume electron microscopy

Job description

In multi-beam electron microscopy (EM), a sample is scanned by tens of beams instead of the single beam in a traditional electron microscope. The increased throughput of multi-beam EM enables large-scale and volume microscopy and identification of rare events or different stages in a dynamic process. In this PostDoc project, the aim is to visualize the life-cycle of Weibel-Palade Bodies (WPBs) in the context of disease using multi-beam EM (FAST-EM) with correlative fluorescence light microscopy of patient-derived endothelial cells.

WPBs are ~1-5 µm-long rod-shaped secretory organelles that store the hemostatic protein Von Willebrand factor (VWF). Following vascular injury endothelial cells (ECs) immediately release large quantities of VWF into the circulation. Low circulating levels of VWF increase the risk of bleeding, such as in Von Willebrand disease, while elevated levels of VWF are associated with elevated risk of thrombosis. The composition and 3D architecture of WPB during fusion and secretion of VWF have thus far remained unclear. This is mainly because traditional electron microscopy approaches have been unable to reliably capture rare events and perform 3D analysis. As a Postdoc operating in a unique collaboration between TU Delft and Erasmus Medical Center, you will tackle this issue with the high-throughput multibeam FAST-EM developed at TU Delft.

You will work at both the Bierings group at Erasmus MC and the Hoogenboom group at TU Delft. You will start by implementing the existing and published FAST-EM array tomography workflow for high-volume high-resolution mapping of WPBs in cultured endothelial cells.

Initial setup will be with endothelial cells (ECFCs) from healthy donors. When successful this will be extended to ECFCs derived from patients, for example with VWF trafficking defects (VWD, Storage pool disease, etc), and to ECs in which specific proteins of interest are depleted via shRNA or CRISPR targeting. You will then capture WPB trafficking events using fluorescence-guided high resolution EM imaging. WPBs and endolysosomal organelles will be labeled using fluorescent reporters or fluorophore-labeled antibodies.

Finally, after successful EM, you will work on segmentation, annotation and analysis of organelles in endothelial cells. A convolutional neural network (based on U-net, for example similar to MitoNet used before on FAST-EM data) will be trained to automatically segment organelles from FAST-EM imaging data, with a focus on WPBs, endolysosomal compartments and vesicles of the ER-Golgi pathway. While you are expected to be actively involved in all aspects of the research, expertise and support for all steps of sample preparation, EM acquisition and data reconstruction, and segmentation is available in both groups via technicians and PhD students on related projects.

Job requirements

You are a highly motivated and talented researcher with a strong interest in electron microscopy for life sciences and the development and application of novel methodology. You enjoy working in a multi-disciplinary environment at the interface of bio-medical research and instrument development and innovation, implementing new techniques to achieve tangible results. Embracing the opportunity to work with leading industrial partners, you know how to collaborate and achieve meaningful results geared to the development of pragmatic applications. You also put your curiosity to good use: you don’t take anomalies for granted and take the initiative to dig deeper and spar with experts. Harnessing your communication skills, you convince colleagues at TU Delft and Delmic of your approach, models and programs. And you enjoy interacting with and coaching talented bachelor and master students, while expanding your own knowledge and competencies.

You also have:

• PhD degree in Molecular Biology, (Applied) Physics, or a closely related field.
• Demonstrable skills in electron microscopy for life sciences, such as cellular EM, volume EM, or cryo-EM.
• A solid understanding and proven expertise in Python and the use of computational techniques, preferably neural networks, for data analysis and segmentation.
• Excellent communication skills, connecting easily with people of diverse scientific backgrounds and being able to bridge research groups at two different locations.
• A proactive and curious mindset, with the ability to explore complex problems independently and collaboratively.
• A good command of spoken and written English, as you’ll be working in an internationally diverse community. You will also write scientific articles and your dissertation in English and participate in English-taught courses.
• An enthusiasm for coaching bachelor, master, and PhD students, and for presenting your work in scientific articles and international conferences.