Project 3

Single-molecule microscopy in zebrafish embryos

Supervisor: Dr. Marcel J.M. Schaaf, co-supervisors: local: Prof. Dr. Annemarie Meijer, external: I. Lyuboshenko (P11)

Objectives:

Capitalising on our recent success at studying individual membrane protein mobility in the skin epidermis of zebrafish embryos using single-molecule microscopy, ESR3 will develop new strategies for protein tagging with the Atto647 dye using the Halo-tag system and extend single molecule microscopy to:

  • non-membrane proteins in cells of the outer epithelial cell layer of the embryos.
  • proteins within leukocytes during mycobacterial infections in the tail fin since it is only a few cell layers thick.

Expected Results:

  • New near-TIRF setup to study the dynamics of non-membrane signalling proteins in outer epithelial cells of the embryos
  • New light sheet and 2-photon microscopy setup to image single molecules (autophagy LC3, chemokines & receptors) in embryos and their dynamics in leukocytes during immune response.

Secondments:

  • academic: P6 ICFO Dr P. Loza, Training in imaging using light sheet microscopy
  • Industrial: P11, Phaseview Dr I.Lyuboshenko, Training in high-speed 3D imaging using light sheet microscopy

Links to other projects: Collaboration with ESR1, 2 & 7 (host pathogen interactions), 1 & 9 (super-resolution) and with ESRs 7 & 8 for image analysis.