Spinning Disk Confocal Microscope
Final Report Abstract
The spinning disk confocal microscopy (SPCM) is important for its ability to quickly obtain 3D images. One of the main area that we have been using the SPCM is to investigate the uptake of nanoparticles in living cells. The nanoparticles can be designed systems for drug delivery, potential environmental hazards or viruses. To determine whether particles have been internalized within the cell, we have developed a novel algorithm, Particle_in_Cell-3D, to quantify the number of nanoparticles within a living cell and, from a 3D multicolor image of the cell, determine whether the observed nanoparticles have been internalized, are in the plasma membrane or are on the cell surface. The functionality of the SPCM with two-cameras, the ability to switch filters quickly and the incubation chamber for live-cell imaging, makes these experiments possible. One important question we are investigating is the toxicity of nanoparticles currently found in the environment. Using this instrument, we showed that platinum containing nanoparticles are quickly taken up by cells and bind to the mitochondria. In another study, we investigated the uptake of silica nanoparticles in HUVEC and HeLa cells. We could show that the HUVEC cells take up the particles 10 times faster and uptake of the silica nanoparticles led to cell death. On the timescale of 10-24 hours, the number of internalized particles was much higher in HeLa cells, even though the initialkinetics were slower. The HeLa cells showed reduced mitochondria activity after nanoparticle uptake but, in constrast to HUVEC cells, no membrane leakage was observed. Besides investigating the toxity of nanoparticles, we also looked at the effectiveness of nano-sized drug delivery systems. We have combined the SPCM with microfluidics to mimic blood flow in living organisms. This could be done as the SPCM has an incubation chamber with enough space to mount the microfluidic device. We could show that binding of nanoparticles by HUH7 cells is dependent on the sheer force. We could also test the specificity of different ligands for targeted uptake of nanoparticles. As another approach for mimicking blood flow conditions, we also used microfluidics with a surface acoustic wave micropump.
Publications
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Uptake Kinetics and Nanotoxicity of Silica Nanoparticles are Cell Type Dependent. Small 9(23)(2013), 3970
Blechinger J. et al.
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Precise quantification of silica and ceria nanoparticle uptake revealed by 3D fluorescence microscopy. Beilstein Journal of Nanotechnology 5 (2014), 1616
Torrano A.A. et al.
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A surface acoustic wave-driven micropump for particle uptake investigation under physiological flow conditions in very small volumes. Beilstein Journal of Nanotechnology 6 (2015), 414
Strobl F.G. et al.
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Assessing potential peptide targeting ligands by quantification of cellular adhesion of model nanoparticles under flow conditions. J Controlled Release 213 (2015), 79
Broda E. et al.
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Cell-Penetrating and Neurotargeting Dendritic siRNA Nanostructures. Angew. Chem. Int. Ed. 53(51) (2015), 1946
Brunner K. et al.
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Dendronized Mesoporous Silica Nanoparticles Provide an Internal Endosomal Escape Mechanism for Successful Cytosolic Drug Release (2015)
Weiss V. et al.
