[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jul 2010 · Journal of Biophotonics
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Apr 2008 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: A variety of physical and biochemical cell properties depends on the function and integrity of the actin cytoskeleton. The actin cytoskeleton mediates crucial cellular functions as migration, intracellular transport, exocytosis, endocytosis, cell stiffness and force generation. 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 or drug effects. Here we applied digital holographic microscopy in the online functional analysis of the actin cytoskeleton changes of a highly differentiated and a dedifferentiated pancreas tumor cell line induced by the marine toxin Latrunculin B. Scanning electron microscopy (SEM) and fluorescence microscopy showed rapid Latrunculin B induced alterations in cell morphology and actin fiber degradation in both pancreatic tumor cell lines. In contrast digital holographic in vivo analysis of the drug dependent dynamic cellular processes revealed unequal changes in cell morphology. While tumor cells with a low metastatic potential showed Latrunculin B induced cell collapse within 4 h the metastatic tumor cells were resistant to Latrunculin B treatment. Spatial resolution of morphological alterations by digital holography detected so far unknown differences in the actin cytoskeleton stability of highly differentiated and dedifferentiated pancreas tumor cell lines. 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.
No preview · Article · Dec 2007 · Medical Laser Application
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Sep 2007 · Journal of Biomedical Optics
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jun 2007 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jun 2007 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: Thin spin-coated films of polystyrene-block-poly(tert-butyl acrylate) (PS690-b-PtBA1210) on various substrates are introduced as versatile, robust reactive platform for the immobilization of (bio)molecules for the fabrication of tailored biointerfaces. The films are characterized by high stability and (bio)reactivity due to the presence of a glassy PS and a reactive PtBA block, respectively. The selective deprotection of the tert-butyl-ester groups in the PtBA skin layer by hydrolysis under acidic conditions, the activation with N-hydroxysuccinimide and the subsequent derivatization with amino functionalized (bio)molecules were investigated. Based on contact angle, FTIR spectroscopy and XPS, fluorescence microscopy and AFM data, it was shown that the (bio)molecules were coupled covalently to the polymer films and that high molecular coverages up to ∼2.4 poly(ethylene glycol) (PEG) molecules per nm2 (Mn = 500 g/mol) were obtained. Organic dyes, polyamidoamine dendrimers, polypeptides, proteins and amino end-functionalized DNA were efficiently and homogeneously immobilized on the PS-PtBA platforms. Grafting of ω-amino functionalized PEG afforded surfaces with substantially reduced non-specific adsorption of proteins and DNA. Owing to the glassy nature of PS and the covalent amide linkages, the derivatized films showed excellent stability under a broad range of processing conditions. Finally, the viability of PS690-b-PtBA1210 platforms as versatile biointerfaces was demonstrated in DNA hybridization experiments, as well as cell-surface interaction studies using pancreatic cancer and K562 cells.
No preview · Article · Jun 2007 · European Polymer Journal
[Show abstract][Hide abstract] ABSTRACT: A new reactive micro-contact printing strategy to fabricate well-defined functional micro- and nanostructure biointerfaces and exploitation of selective block copolymer films by using a volatile and highly diffusive reactant trifluroacetic acid (TFA) was analyzed. The reactive tert-butyl ester moieties at the film surface and microphase-separated glassy polyesterene domains in the film interior was used as a block copolymer film, that provided thermal and processing stability. The fluorescence microscopy images reveals the zero coverage of fluorescinamine in the nonhydrolyzed areas and polymer film deformation by capillary forces. The Atomic Force Microscopic (AFM) studies of TFA stamps shows that the binary cell-adhesive/cell-repulsive nanopatterns affect the cancer cell morphology. The line-edge definition observed in the friction-mode AFM images between hydrophilic and hydrophobic areas show the occurrence of a laterally homogeneous and well-defined deprotection polymer reaction.
No preview · Article · Jan 2007 · Advanced Materials
[Show abstract][Hide abstract] ABSTRACT: 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 cell's cytoskeleton. The results demonstrate that digital holographic microscopy is a quantitative phase contrast technique for living cells under conventional laboratory conditions.
No preview · Article · May 2006 · Journal of Biomedical Optics
[Show abstract][Hide abstract] ABSTRACT: A modular digital holographic microscopy system for integration in commercial microscopes has been developed. The reconstruction of the digitally captured holograms is performed by application of a spatial phase shifting non diffractive reconstruction method. As a consequence of the applied algorithms the reconstructed holographic images contain not the disturbing terms "twin image" and "zero order". In combination with microscope lenses the system's lateral resolution is improved up to the diffraction limit. Digital holographic focus adjustment with constant imaging scale allows multi focus imaging of object planes and subsequent focus correction from only a single captured hologram. Results of investigations on technical specimen characterize the lateral and axial resolution of the system. The applicability of the system is demonstrated by results obtained from drug stimulated cellular morphology changes and apoptosis monitoring of living pancreas cells.
No preview · Article · Apr 2006 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: Oxygen preference of the planktonic crustacean Daphnia magna was observed within an oxygen gradient. Long-term acclimation to specific oxygen conditions determined the preferred oxygen tension. Animals from hypoxic acclimation stayed in oxygen-poor water at first. They chose oxygen tensions above the critical partial pressure, thus maintaining aerobic energy supply, but they avoided higher oxygen concentrations. However, they gradually took more oxygen-rich positions within 2 d. Animals from severe hypoxic and hyperoxic acclimation showed stenoxic behavior. Specimens raised in moderately hypoxic or normoxic medium spread out more widely in the oxygen gradient (euryoxic behavior). The possible role of hemoglobin and antioxidant defense capacity limiting the animals' distribution at low or high oxygen concentrations, respectively, is discussed. In tests with mixed groups of D. magna from opposing oxygen acclimation conditions, the animals clustered at intermediate oxygen conditions. However, the acclimation condition of the major group was found to determine the position taken within the gradient to a greater extent. Selected oxygen tensions were closer to the preference values of the larger group, yet the influence of the minority prevented the majority in heterogeneous groups from taking their originally determined preference position. Thus, aggregation behavior interfered with the acclimation-dependent oxygen preference behavior.
No preview · Article · May 2005 · Physiological and Biochemical Zoology