Lichtscheibenmikroskopie und Ratio Imaging - Entwicklung von Modulen und deren Anwendung zur Untersuchung von Tumorzellsphäroiden

The aim of this work was the combination of ratio imaging and light sheet fluorescence microscopy for examination of the redox state in tumor cell spheroids using the redox sensitive green fluorescent protein Grx1-roGFP2. This sensor mirrors the redox state of glutathione and is characterized by different excitation spectra in the reduced and oxidized state, whereas the emission spectra are identical. In the scope of this work add-on modules for an existing inverted microscope were developed. The SPIM (Single Plane Illumination Microscopy) module mounted to the fixed part of the positioning stage of the microscope allows for simultaneous light sheet illumination of the sample with two different wavelengths. Samples are located in rectangular glass capillaries placed in special capillary holders adapted to the movable part of the positioning stage. The capillaries can be attached to a fluidic system providing the sample with agents during the measurement. The module for ratio imaging is mounted to a side port of the microscope. Its main components are a field aperture, a beam splitter, an image transmitting fiber bundle and a time-gated camera synchronized with two pulsed laser diodes (shifted in time against each other) for excitation. It thus allows for simultaneous recording of two fluorescence images generated by different excitation wavelengths each on one side of the camera’s sensor although the fluorescence emission is in the same spectral range for both excitation wavelengths. Validation was performed by imaging changes of the redox state in tumor cell spheroids induced by hydrogen peroxide or staurosporine-induced apoptosis. Furthermore fluorescence lifetime imaging in single layers of cell spheroids was performed with light sheet illumination, and z-stacks of cell spheroids are presented to demonstrate the possibilities opened up by the SPIM module and corresponding sample holding

Monitoring of Apoptosis in 3D Cell Cultures by FRET and Light Sheet Fluorescence Microscopy

Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure