| dc.description.abstracteng | In this project, a promising imaging method using NIR fluorescent, DNA-wrapped CNTs is applied to study their optical and dynamical behavior in syncytial Drosophila embryos.
Drosophila (commonly known as vinegar fly) is one of the most studied model organisms in developmental biology and increasingly draws attention from physical sciences.
In hours-long syncytial stage of embryonic development (embryogenesis), nuclei in embryonic Drosophila form a highly dynamic 2D cortical layer unveiling a multitude of interesting dynamics.
Capturing details of microscopic mechanics during embryogenesis on short time scales during such long measurement times pushes demands for both single-molecule and single-nanoparticle fluorescence experiments to their boundaries.
Semiconducting NIR fluorescent CNTs are promising novel fluorescent markers for in vivo studies, since they have unique optoelectronic properties.
They display extraordinary photostability and Stokes shifts that can reach several hundred nanometers, having an extended excitation spectrum in the VIS range.
The photostable and intermittency-free NIR fluorescence of CNTs enable us to capture high frequency information of individual CNT trajectories in the living embryos, making CNTs valuable probes for long-time tracking over multiple division cycles inside living Drosophila embryos.
We solubilize the hydrophobic CNTs in watery solutions and use biochemical linking methods to potentially assess specific binding of fluorescent CNTs to single kinesin-5 molecules in transgenic Drosophila embryos.
With microinjection we introduce DNA-wrapped CNTs into syncytial Drosophila embryos of two transgenic types. Here, we present a custom-built setup, allowing simultaneous imaging of CNT NIR fluorescence and EGFP-tagged nuclear histones.
With an IR spectrometer integrated into this VIS and NIR wide-field fluorescence microscope setup, we characterize the excitation spectrum of the CNTs used in our experiments to find the optimal wavelength for excitation of CNT NIR fluorescence.
During measurements, we combine high frequency CNT NIR signals with corresponding low frequency nuclear His-EGFP signals.
This combination of two imaging channels provides a powerful tool for conducting single-nanoparticle experiments in vivo with CNTs correlated with EGFP labeling in the VIS channel over a wide time range, enabling us to simultaneously capture intracellular dynamics on multiple time scales.
Within each of these embryos, we observe individual CNT fluorescence signals and VIS fluorescence of EGFP in nuclear histones during various division cycle phases in the cortical layer.
We superimpose these fluorescence signals measured in different transgenic types of Drosophila for qualitative spatio-temporal orientation.
Furthermore, we analyze dynamics of functionalized CNTs in the cortex of living embryos of different transgenic Drosophila.
Information about intracellular dynamics of CNTs in these fly types is obtained by single-nanoparticle tracking and subsequent correlation analyses of individual CNTs.
From these results, we infer dominant diffusive and sub-diffusive behavior of CNTs in the investigated embryos and find the hydrodynamic length of CNTs.
Furthermore, we demonstrate quantitative results obtained from systematic imaging in embryonic tissue.
In addition, we use NIR fluorescent CNTs in conjunction with particle image velocimetry to capture mesoscopic bulk dynamics of the cytoplasmic flow in developing Drosophila embryos. | de |