Veröffentlichungen

For many biomedical applications, laser-assisted methods are essential to enhance the three-dimensional (3D) resolution of a light microscope. In this report, we review possibilities to improve the 3D imaging potential by axial tomography. This method allows us to rotate the object in a microscope into the best perspective required for imaging. Furthermore, images recorded under variable angles can be combined to one image with isotropic resolution. After a brief review of the technical state of the art, we show some biomedical applications, and discuss future perspectives for Deep View Microscopy and Molecular Imaging.

Optical tweezers are based on a transfer of momentum from laser photons to a transparent particle and are often applied to hold, move or manipulate single living cells. This paper discusses up to which light dose irradiance can be regarded as non-phototoxic, summarizes some possible applications, and describes experimental setups, which might fulfil these requirements.

Methods and applications of light microscopy in the life sciences are compared with respect to 3D imaging, resolution, light exposure, sensitivity and recording time. While conventional wide-field or laser scanning microscopy appear appropriate for smaller samples of only a few micrometers size with a limited number of light exposures, light sheet microscopy appears to be an optimal method for larger 3D cell cultures, biopsies or small organisms, if multiple exposures or long measuring periods are desired. Super-resolution techniques should be considered in the context of high light exposure possibly causing photobleaching and photo-toxicity to living specimens.

Here we present a simple and robust experimental setup for super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) and exchangeable objective lenses for epi-illumination and total internal reflection fluorescence (TIRF) microscopy. To show the applicability of this approach, both methods are used to measure translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane upon stimulation by insulin or insulin-mimetic compounds with a lateral resolution around 100 nm and an axial resolution around 200 nm.

The present manuscript gives a short overview on Förster Resonance Energy Transfer (FRET) of molecular interactions in the nanometre range. First, its principle is described and a short historical overview is given. Subsequently some principal methods and applications of FRET sensing and imaging are described (with some emphasis on fluorescence lifetime imaging, FLIM), and finally two innovative FRET techniques are presented in more detail. Applications are focused on measurements of living cells.

Laser microbeam techniques are presented, which permit the introduction of molecules or small particles into living cells. Possible mechanisms - including photochemical, photothermal and opto-mechanical interactions (ablations) - are induced by continuous wave (cw) or pulsed lasers of different wavelength, power and mode of operation. Laser-assisted optoporation permits the uptake of fluorescent dyes as well as DNA plasmids for cell transfection, and, in addition to its broad application to cultivated cells, may have some clinical potential.

In the context of various approaches for super-resolution microscopy Structured Illumination Microscopy (SIM) offers several advantages: it needs rather low light doses (with a low risk of phototoxicity or photobleaching), is comparably fast and flexible concerning the use of microscopes, objective lenses and cameras, and has a potential for 3D imaging. This paper describes an experimental setup for SIM with first diffraction orders of a spectral light modulator (SLM) creating an interference pattern in two dimensions. We kept this system rather compact with a comparably large illuminated object field, validated it with nano-beads and applied it further to living cells for imaging the cytoskeleton, mitochondria or cell nuclei with a resolution slightly above 100 nm. Its advantages, challenges and limitations – concerning cameras, acquisition time, depth of imaging, light exposure, and combination with further super-resolving methods - are discussed.

An experimental setup for super-resolutionmicroscopy by structured illumination is presented, preliminary experiments of nano-beadsand living cells with a resolution around 100 nm are described, andfurther requirements for live cell microscopy are discussed.

Previously we reported on the uptake and interaction of cytotoxic doxorubicin in MCF-7 breast cancer cells grown as standard 2-dimensional cell cultures. Now improved experimental techniques – including axial tomography and Light Sheet Fluorescence Microscopy (LSFM) – permit observation of single cells from any side as well as detection of individual layers in multi-cellular spheroids. Therefore, uptake of doxorubicin in the cell nucleus as well re-localization in the cytoplasm at longer incubation times is well documented. Based on a calcein-AM test we could prove high cytotoxicity in 3D cell cultures at 48-96h after incubation. Simultaneously disintegration of cell spheroids and formation of a degradation product became obvious. Fluorescence lifetime imaging microscopy (FLIM) is presently used to distinguish the fluorescence of doxorubicin and its degradation product, and Structured Illumination Microscopy (SIM) is suggested to improve resolution down to about 100 nm.

Two types of laser illumination in live cell microscopy with a focus on the sample or in the aperture plane of the microscope objective lens are distinguished. For the second case two examples are described, namely light scattering microscopy with angular resolution and Structured Illumination Microscopy (SIM) with two interfering laser beams. Appropriate applications include morphological studies of cells undergoing apoptosis and mitochondrial imaging with increased resolution.