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Visualising thrombus formation in 4D – Application of state-of-the-art light sheet microscopy

Principal Supervisor: Dr Steve Thomas - Institute of Cardiovascular Sciences

Co-supervisor: Professor Steve Watson, Dr Malou Zuidscherwoude

PhD project title: Visualising thrombus formation in 4D – Application of state-of-the-art light sheet microscopy

University of Registration: University of Birmingham

Project outline:

The project will involve two newly developed forms of light sheet microscopy (diSPIM and lattice) to record the events that underlie thrombus formation in 4-dimensions. The lattice light sheet was developed by Eric Betzig who was awarded the Nobel Prize in 2014. No other university in Europe currently has a lattice light sheet system.

Thrombus formation is a fundamental process by which animals are able to respond to injury to prevent blood loss and to promote wound repair. In mammals, platelets are the blood cells that play a critical role this process. The formation of a thrombus is a complex process that involves dynamic interactions between endothelial cells and sub-endothelial matrix proteins of the vessel wall, circulating platelets & leukocytes, and the coagulation cascade. It occurs in 3D over time (i.e. 4D) and is influenced by a range of receptors, signalling molecules, plasma components and blood flow. Recent work using intra-vital microscopy has established that individual thrombi are heterogeneous structures which consist of various regions including an inner core of P-selectin positive fully activated platelets which support fibrin formation and an outer shell of loosely adherent platelets which are either resting or only partially activated1. This heterogeneity reflects the spatial and temporal nature of platelet activation during thrombus formation and requires coordination of signalling events. To understand the basic biological processes at play during thrombus growth and regulation we need to visualise them occurring in real time. However, this is technically challenging due to the speed and complexity of the process. A variety of methods have been used to image thrombus formation including in vitro flow studies using glass capillaries or biochips to enable dissection and characterisation of the events that occur in the growing thrombus, through to animal models where thrombus formation can be induced in exteriorised tissue via physical damage to the blood vessel. Whilst these methods are widely used the microscopy methods used for imaging these events can be the “rate-limiting” step due to limited imaging depth, capture speed or photo-damage caused by high light intensities.

Light sheet fluorescence microscopy (LSFM) uses 2 objective lenses placed at 90° to each other. One objective illuminates the sample with a lightsheet whilst the other collects fluorescence emission2,3. This has advantages over standard fluorescence including low photo-toxicity and the ability to rapidly scan the sheet through the sample. It is therefore ideal for real time studies where 3D volumes can be collected over time to enable complex biological processes to be studied. We have recently acquired state-of–the-art diSPIM and lattice light sheet microscopes and therefore the aim of this project is to apply LSFM to studying thrombus formation in real time, in order to increase our understanding of this fundamental mammalian response to tissue injury.

The student will be based in the Birmingham Platelet Group at the University of Birmingham medical school and will learn cellular and molecular techniques for studying platelet biology as well as using state-of-the-art imaging technology to address fundamental biological questions regarding platelet activation and thrombus formation. We will use and develop LSFM to image these events and ask questions such as “what is the role of the cytoskeleton in the adhesion of platelets under flow conditions?” and “how is thrombus formation affected by agents that block specific pathways of platelet activation?” This work will advance our understanding of the biology and dynamic nature of this fundamental mammalian process.

References:

  1. Ivancui & Stalker 2015, JTH doi: 10.1111/jth.13145
  2. Wu et al 2011, PNAS 108 (43) 17708–17713
  3. Chen et al 2014, Science. DOI: 10.1126/science.1257998

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • Platelet preparation from both mouse and humans
  • Platelet function assays
  • In vitro flow adhesion assays (including but not limited to Cellix Biochips and capillary based methods)
  • Advanced fluorescence microscopy including Light sheet, TIRF and confocal imaging
  • Development of protocols for appropriate sample presentation for light sheet imaging including 3D printing
  • Computational image analysis

Contact: Dr Steve Thomas, University of Birmingham