dc.description.abstracteng | A developing embryo is a striking and fascinating example of self-organization and
collective behavior of biological matter. In the presented work, I investigated the
cytoplasmic interior of Drosophila melanogaster embryos. In its early stage the
embryo forms a syncytium, i.e. multiplying nuclei are not yet separated by cell
membranes, but are interconnected by cytoskeletal polymer networks consisting of
actin and microtubules. Between division cycles 9 and 13, nuclei form a 2D cortical
layer with the cytoskeleton associated to it. To probe the mechanical properties
and dynamics of this self-organizing "pre-tissue", I measured shear moduli in the
embryo by high-speed video microrheology. Therefore, I built a multi-color fluores-
cence microscope for simultaneously imaging at high speeds with frame rates of up
to several kHz and at normal video rates in order to access an extended frequency
range. I recorded position fluctuations of injected micron-sized fluorescent beads
and characterized the viscoelasticity of the embryo in different locations. Thermal
fluctuations dominated over non-equilibrium activity for frequencies between 0.3 and
1000 Hz. Between nuclear layer and central yolk the cytoplasm was homogeneous
and viscously-dominated, with a viscosity three orders of magnitude higher than
that of water. Close to the nuclear layer, particularly close to the cortex, I found an
increase of the elastic and viscous moduli consistent with an increased microtubule
density. Mechanical response near the nuclear layer is likely to be caused by loosely
entangled microtubule networks, whereas in the interior, towards the central yolk,
it is due to a macromolecular solution. Drug-interference experiments showed that
microtubules contribute to the measured viscoelasticity inside the embryo, whereas
actin only plays a minor role in the regions I probed with the micron sized beads, i.e.
outside of the actin caps and cortex. Measurements at different stages of the nuclear
division cycle showed little variation. During nuclear separation at anaphase I found
directed motion of probe particles, the only measurable sign for non-equilibrium ac-
tivity, so far.
Secondly I investigated single-walled carbon nanotubes (CNT) as fluorescent and
trappable probes by means of the custom-built setup incorporating near-infrared
imaging and spectroscopy instruments as well as multiple trapping lasers and an
interferometric detection system. CNTs have an intrinsic near-infrared fluorescence,
emitting within a wavelength window almost free of autofluorescence in biological
tissue. Hence, imaging of single CNTs injected into the whole living fly embryo by
wide-field microscopy was possible. Mean squared displacements of tracked CNTs
within the embryonic cytoplasm showed diffusive and subdiffusive motion as well as
directed movements, some of them correlating with nuclear motion. In vitro experi-
ments characterized CNTs as fluorescent probes, lacking fluorescence intermittency,
exhibiting short fluorescence cycle lifetimes and high photostability (though lower
than in previous findings). I optically trapped CNTs, simultaneously confirmed
by fluorescence microscopy and interferometric detection. The shape of a position
power spectral density of a trapped CNT was close to a Lorentzian and the mea-
surement of the position variance allowed a determination of the number of trapped
particles. No clear signs for resonance effects on trapping efficiency were observed. | de |