Ilona Bredebusch

Investigation of living pancreas tumor cells by digital holographic microscopy.

Digital holographic microscopy provides new facilities for contactless and marker-free quantitative phase contrast imaging. In this work, a digital holographic microscopy method for the integral refractive index determination of living single cells in cell culture medium is presented. Further, the obtained refractive index information is applied to full field thickness and shape determination of adherent pancreas tumor cells, as well as for analysis of drug-induced dynamic changes of a single cells cytoskeleton. The results demonstrate that digital holographic microscopy is a quantitative phase contrast technique for living cells under conventional laboratory conditions.

Integral refractive index determination of living suspension cells by multifocus digital holographic phase contrast microscopy

A method for the determination of the integral refractive index of living cells in suspension by digital holographic microscopy is described. Digital holographic phase contrast images of spherical cells in suspension are recorded, and the radius as well as the integral refractive index are determined by fitting the relation between cell thickness and phase distribution to the measured phase data. The algorithm only requires information about the refractive index of the suspension medium and the image scale of the microscope system. The specific digital holographic microscopy advantage of subsequent focus correction allows a simultaneous investigation of cells in different focus planes. Results obtained from human pancreas and liver tumor cells show that the integral cellular refractive index decreases with increasing cell radius.

Inverted microcontact printing on polystyrene-block-poly(tert-butyl acrylate) films: a versatile approach to fabricate structured biointerfaces across the length scales.

The combination of the recently introduced soft lithographic technique of inverted microcontact printing (i-muCP) and spin-coated films of polystyrene- block-poly( tert-butyl acrylate) (PS 690- b-P tBA 1210) as a reactive platform is shown to yield a versatile approach for the facile fabrication of topographically structured and chemically patterned biointerfaces with characteristic spacings and distances that cross many orders of magnitude. The shortcomings of conventional muCP in printing of small features with large spacings, due to the collapse of small or high aspect ratio stamp structures, are circumvented in i-muCP by printing reactants using a featureless elastomeric stamp onto a topographically structured reactive polymer film. Prior to molecular transfer, the substrate-supported PS 690- b-P tBA 1210 films were structured by imprint lithography resulting in lateral and vertical feature sizes between >50 microm-150 nm and >1.0 microm-18 nm, respectively. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and water contact angle measurements provided evidence for the absence of surface chemical transformations during the imprinting step. Following the previously established hydrolysis and activation protocol with trifluoroacetic acid and N-hydroxysuccinimide, amino end-functionalized poly(ethylene glycol) (PEG-NH 2), as well as bovine serum albumin and fibronectin as model proteins, were successfully transferred by i-muCP and coupled covalently. As shown, i-muCP yields increased PEG coverages and thus improved performance in suppressing nonspecific adsorption of proteins by exploiting the high local concentrations in the micro- and nanocontacts during molecular transfer. The i-muCP strategy provides access to versatile biointerface platforms patterned across the length scales, as shown for guided cancer cell adhesion, which opens the pathway for systematic cell-surface interaction studies.

Monitoring of laser micromanipulated optically trapped cells by digital holographic microscopy

For a precise manipulation of particles and cells with laser light as well as for the understanding and the control of the underlying processes it is important to visualize and quantify the response of the specimens. Thus, we investigated if digital holographic microscopy (DHM) can be used in combination with microfluidics to observe optically trapped living cells in a minimally invasive fashion during laser micromanipulation. The obtained results demonstrate that DHM multi-focus phase contrast provides label-free quantitative monitoring of optical manipulation with a temporal resolution of a few milliseconds.

Techniques and Applications of Digital Holographic Microscopy for Life Cell Imaging

In connection with microscopy, digital holography provides contact-less, marker-free, quantitative phase-contrast imaging. Particularly, the feature of (subsequent) numerical auto focus adjustment enables applications in the field of life cell analysis. Here, prospects for long term time-lapse investigations in toxicology and cancer research as well as for monitoring of fast dynamic processes like shape variations are opened up. The evaluation of the obtained quantitative phase contrast provides data for thickness monitoring as well as for the observation of cell swelling kinetics due to osmotic stimulation. Results from investigations on living cells demonstrate digital holographic microscopy application fields for quantitative life cell analytics.

Determination of the integral refractive index of cells in suspension by digital holographic phase contrast microscopy

A digital holographic microscopy method for the determination of the integral refractive index of living cells in suspension is presented. Therefore, digital holographic phase contrast images of trypsinized cells in suspension are recorded. Afterwards, the cell radius and the integral cellular refractive index are determined by fitting of a two dimensional model. The applied algorithm requires only information about the refractive index of the suspension medium and the scale of the microscopic imaging system. The fitting algorithm is characterized on simulated phase data and digital holographic phase contrast images of beads. Results obtained from living human pancreas tumor cells demonstrate the applicability of the method for cell characterization.

Dynamic in vivo analysis of drug induced actin cytoskeleton degradation by digital holographic microscopy

The actin cytoskeleton mediates a variety of crucial cellular functions as migration, intracellular transport, exocytosis, endocytosis and force generation. The highly dynamic actin fibers are therefore targets for several drugs and toxins. However the study of actin interfering processes by standard microscopy techniques fails in the detailed resolution of dynamic spatial alterations required for a deeper understanding of toxic effects. Here we applied digital holographic microscopy in the online functional analysis of the actin cytoskeleton disrupting marine toxin Latrunculin B. SEM and fluorescence microscopy showed rapid Latrunculin B induced alterations in cell morphology and actin fiber degradation in pancreas tumor cells. The dynamic digital holographic in vivo analysis of the drug dependent cellular processes demonstrated differences in the actin cytoskeleton stability of highly differentiated and dedifferentiated pancreas tumor cell lines. The spatial resolution of the morphological alterations revealed unequal changes in cell morphology. While cells with a low metastatic potential showed Latrunculin B induced cell collapse within 4 h the metastatic tumor cells were increased in cell volume indicating Latrunculin B effects also on cell water content. These data demonstrate that marker free, non-destructive online analysis of cellular morphology and dynamic spatial processes in living cells by digital holography offers new insights in actin dependent cellular mechanisms. Digital holographic microscopy was shown to be a versatile tool in the screening of toxic drug effects and cancer cell biology.