Session Title: Imaging III
Session Date/Time: Saturday 19/09/2015 | 16:30-18:00
Paper Time: 17:50
First Author: : N.Celik GERMANY
Co Author(s): : G. Best F. Schock K. Licha G. Auffarth C. Cremer S. Dithmar
PURPOSE:To visualize interaction between Bevacizumab and retinal pigment epithelium in vitro
Department of Ophthalmology, University of Heidelberg
A specialized microscope which combines Structured Illumination Microscopy (SIM) and Spectral Precision Determination Microscopy (SPDM) was constructed (laser excitation wavelengths 488, 568 and 647 nm), which enabels highly resolved 3D-SIM images with a resolution of down to 150 nm and localization of fluorophors at a high accuracy of 20 nm. Transiently transfected ARPE-19 cells were used where GFP (green fluorescent protein) was targeted against the nucleus and RFP (red fluorescent protein) against the plasma membrane. Confluent cultured ARPE-19 cells were treated with labeled Bevacizumb (6s-IDCC) in clinically relevant concentrations. Drug uptake was examined with superresolution microscopy.
Uptake and intracellular accumulation of labeld Bevacizumab in ARPE-19 cells could be observed. Multichannel 3D-SIM allows the determination of drug distribution inside and outside of cells without apparent harm to the living cell. Using higher light intensities, SPDM allows an exact localization of single drug molecules.
For the first time we show the implementation of a superresolution microscope for living RPE cells. The here presented combination of SIM and SPDM enables visualization of drug uptake and intracellular behavior. By the use of the two techniques, single drug molecules can be localized with a precision about 10 fold better than the conventional diffraction limit.