Multiview light-sheet microscope for rapid in toto imaging.
Krzic, U., Gunther, S., Saunders, T.E., Streichan, S.J. & Hufnagel, L.
Nat Methods. 2012 Jun 3;9(7):730-3. doi: 10.1038/nmeth.2064.
We present a multiview selective-plane illumination microscope (MuVi-SPIM), comprising two detection and illumination objective lenses, that allows rapid in toto fluorescence imaging of biological specimens with subcellular resolution. The fixed geometrical arrangement of the imaging branches enables multiview data fusion in real time. The high speed of MuVi-SPIM allows faithful tracking of nuclei and cell shape changes, which we demonstrate through in toto imaging of the embryonic development of Drosophila melanogaster.
Quantitative fluorescence imaging of protein diffusion and interaction in living cells.
Capoulade, J., Wachsmuth, M., Hufnagel, L. & Knop, M.
Nat Biotechnol. 2011 Aug 7;29(9):835-9. doi: 10.1038/nbt.1928.
Diffusion processes and local dynamic equilibria inside cells lead to nonuniform spatial distributions of molecules, which are essential for processes such as nuclear organization and signaling in cell division, differentiation and migration. To understand these mechanisms, spatially resolved quantitative measurements of protein abundance, mobilities and interactions are needed, but current methods have limited capabilities to study dynamic parameters. Here we describe a microscope based on light-sheet illumination that allows massively parallel fluorescence correlation spectroscopy (FCS) measurements and use it to visualize the diffusion and interactions of proteins in mammalian cells and in isolated fly tissue. Imaging the mobility of heterochromatin protein HP1alpha (ref. 4) in cell nuclei we could provide high-resolution diffusion maps that reveal euchromatin areas with heterochromatin-like HP1alpha-chromatin interactions. We expect that FCS imaging will become a useful method for the precise characterization of cellular reaction-diffusion processes.
Collective cell migration guided by dynamically maintained gradients.
Streichan, S.J., Valentin, G., Gilmour, D. & Hufnagel, L.
Phys Biol. 2011 Aug;8(4):045004. doi: 10.1088/1478-3975/8/4/045004. Epub 2011 Jul12.
How cell collectives move and deposit subunits within a developing embryo is a question of outstanding interest. In many cases, a chemotactic mechanism is employed, where cells move up or down a previously generated attractive or repulsive gradient of signalling molecules. Recent studies revealed the existence of systems with isotropic chemoattractant expression in the lateral line primordium of zebrafish. Here we propose a mechanism for a cell collective, which actively modulates an isotropically expressed ligand and encodes an initial symmetry breaking in its velocity. We derive a closed solution for the velocity and identify an optimal length that maximizes the tissues' velocity. A length dependent polar gradient is identified, its use for pro-neuromast deposition is shown by simulations and a critical time for cell deposition is derived. Experiments to verify this model are suggested.