Project 2

Correlative light and electron microscopy in the mycobacteria-infected zebrafish embryo

Supervisor: Prof. Dr Annemarie Meijer, co-supervisors: local: Marcel Schaaf, external: Jochen Gehrig (P9)

Objectives:

The zebrafish embryo is a well-established model to study how tuberculosis hides in subcellular compartments of immune cells and induces infected leukocytes to form organised cellular structures, such as granulomas, the hallmark structures of mycobacterial infections and key to understand the pathogenesis of tuberculosis. Aims are:

  • Image immune defences & subcellular mycobacterial localisation during granuloma formation using our VAST BioImager – Confocal setup designed for automated handling and high-resolution imaging of large numbers of sedated zebrafish larvae.
  • Correlate VAST/confocal imaging with block face scanning electron microscopy (SEM) for ultrastructural visualisation

Expected Results:

  • 3D reconstruction of activated immune responses during mycobacterial granuloma formation using available zebrafish transgenic reporter lines for host defence mechanisms (autophagy (LC3-GFP) and cytokine gene (TNF, IL1B) expression) as well as new reporters (chemokine receptor signalling, Ca2+ signalling) to be developed in WP2.
  • Correlative analysis of VAST/confocal & block face SEM data to define subcellular compartments where mycobacteria reside when different host defence signalling pathways are activated.

Secondments:

  • Academic: P1, UM Dr G Lutfalla; New reporter lines (from WP2) & sample collection for block face SEM.
  • Industrial: P9 Acquifer AG, Dr Jochen Gehrig; Developing an automated image analysis pipeline for data acquired with the VAST Biolmager platform.

Links to other projects: Collaboration with ESR 1, 3 & 7 (host pathogen interactions), 9 (incubation chambers & microscope setups) and ESR12 for industrial applications.