Steffi Ketelhut

Automated three-dimensional tracking of living cells by digital holographic microscopy.

Digital holographic microscopy (DHM) enables a quantitative multifocus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms enables subsequent automated focus realignment by numerical propagation of the digital holographically reconstructed object wave. In combination with a calibrated optical imaging system, the obtained propagation data quantify axial displacements of the investigated sample. The evaluation of quantitative DHM phase contrast images also enables an effective determination of lateral cell displacements. Thus, 3-D displacement data are provided. Results from investigations on sedimenting red blood cells and HT-1080 fibrosarcoma cells in a collagen tissue model demonstrate that DHM enables marker-free automated quantitative dynamic 3-D cell tracking without mechanical focus adjustment.

Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

Quantitative Stain-Free and Continuous Multimodal Monitoring of Wound Healing In Vitro with Digital Holographic Microscopy

Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure are commonly evaluated in in vitro wound assays. However, staining procedures and discontinuous monitoring are major drawbacks hampering accurate assessment of wound assays. We therefore investigated digital holographic microscopy (DHM) to appropriately monitor wound healing in vitro and secondly, to provide multimodal quantitative information on morphological and functional cell alterations as well as on motility changes upon cytokine stimulation. Wound closure as reflected by proliferation and migration of Caco-2 cells in wound healing assays was studied and assessed in time-lapse series for 40 h in the presence of stimulating epidermal growth factor (EGF) and inhibiting mitomycin c. Therefore, digital holograms were recorded continuously every thirty minutes. Morphological changes including cell thickness, dry mass and tissue density were analyzed by data from quantitative digital holographic phase microscopy. Stimulation of Caco-2 cells with EGF or mitomycin c resulted in significant morphological changes during wound healing compared to control cells. In conclusion, DHM allows accurate, stain-free and continuous multimodal quantitative monitoring of wound healing in vitro and could be a promising new technique for assessment of wound healing.

Chitosan encapsulation modulates the effect of capsaicin on the tight junctions of MDCK cells

Capsaicin has known pharmacological effects including the ability to reversibly open cellular tight junctions, among others. The aim of this study was to develop a strategy to enhance the paracellular transport of a substance with low permeability (FITC-dextran) across an epithelial cell monolayer via reversible opening of cellular tight junctions using a nanosystem comprised by capsaicin and of chitosan. We compared the biophysical properties of free capsaicin and capsaicin-loaded chitosan nanocapsules, including their cytotoxicity towards epithelial MDCK-C7 cells and their effect on the integrity of tight junctions, membrane permeability and cellular uptake. The cytotoxic response of MDCK-C7 cells to capsaicin at a concentration of 500 μM, which was evident for the free compound, is not observable following its encapsulation. The interaction between nanocapsules and the tight junctions of MDCK-C7 cells was investigated by impedance spectroscopy, digital holographic microscopy and structured illumination fluorescence microscopy. The nanocapsules modulated the interaction between capsaicin and tight junctions as shown by the different time profile of trans-epithelial electrical resistance and the enhanced permeability of monolayers incubated with FITC-dextran. Structured illumination fluorescence microscopy showed that the nanocapsules were internalized by MDCK-C7 cells. The capsaicin-loaded nanocapsules could be further developed as drug nanocarriers with enhanced epithelial permeability.

Sensing dynamic cytoplasm refractive index changes of adherent cells with quantitative phase microscopy using incorporated microspheres as optical probes

Abstract. The intracellular refractive index is an important parameter that describes the optical density of the cytoplasm and the concentration of the intracellular solutes. The refractive index of adherently grown cells is difficult to access. We present a method in which silica microspheres in living cells are used to determine the cytoplasm refractive index with quantitative phase microscopy. The reliability of our approach for refractive index retrieval is shown by data from a comparative study on osmotically stimulated adherent and suspended human pancreatic tumor cells. Results from adherent human fibro sarcoma cells demonstrate the capability of the method for sensing of dynamic refractive index changes and its usage with microfluidics.

Simple and fast spectral domain algorithm for quantitative phase imaging of living cells with digital holographic microscopy.

We present a simple and fast phase aberration compensation method in digital holographic microscopy (DHM) for quantitative phase imaging of living cells. By analyzing the frequency spectrum of an off-axis hologram, phase aberrations can be compensated for automatically without fitting or pre-knowledge of the setup and/or the object. Simple and effective computation makes the method suitable for quantitative online monitoring with highly variable DHM systems. Results from automated quantitative phase imaging of living NIH-3T3 mouse fibroblasts demonstrate the effectiveness and the feasibility of the method.

Enhanced quantitative phase imaging in self-interference digital holographic microscopy using an electrically focus tunable lens.

Self-interference digital holographic microscopy (DHM) has been found particular suitable for simplified quantitative phase imaging of living cells. However, a main drawback of the self-interference DHM principle are scattering patterns that are induced by the coherent nature of the laser light which affect the resolution for detection of optical path length changes. We present a simple and efficient technique for the reduction of coherent disturbances in quantitative phase images. Therefore, amplitude and phase of the sample illumination are modulated by an electrically focus tunable lens. The proposed method is in particular convenient with the self-interference DHM concept. Results from the characterization of the method show that a reduction of coherence induced disturbances up to 70 percent can be achieved. Finally, the performance for enhanced quantitative imaging of living cells is demonstrated.

Multimodal label-free in vitro toxicity testing with digital holographic microscopy

Common in vitro toxicity tests of drugs, chemicals or nanomaterials involves the measurement of cellular endpoints like stress response, cell viability, proliferation or cell death. The assay systems determine enzyme activity or protein expression by optical read out of enzyme substrates or marker protein labeling. These standard procedures have several disadvantages. Cellular processes have to be stopped at a distinct time point for the read out, where usually only parts of the cells were affected by the treatment with substances. Typically, only one parameter is analyzed and detection of cellular processes requires several time consuming incubations and washing steps. Here we have applied digital holographic microscopy (DHM) for a multimodal label-free analysis of drug toxicity. NIH 3T3 cells were incubated with 1 μM Taxol for 24 h. The recorded quantitative phase images were analyzed for cell thickness, cell volume, dry mass and cell migration. Taxol treated cells showed rapidly decreasing cell motility as measure of cell viability. A short increase in cell thickness and dry mass indicated cell division and growth in control cells, whereas Taxol treatment resulted in a continuous increase in cell height followed by a rapid decrease and a decrease of dry mass as indicators of cell death. Multimodal DHM analysis of drug treatment by multiple parameters allows direct and label-free detection of several toxicity parameters in parallel. DHM can quantify cellular reactions minimally invasive over a long time period and analyze kinetics of delayed cellular responses. Our results demonstrate digital holographic microscopy as a valuable tool for multimodal toxicity testing.

Optical tweezers induced photodamage in living cells quantified with digital holographic phase microscopy

Optical tweezers are a versatile technique to manipulate living biological specimen in a contact-less way. The interaction with living cells can be performed, for example, through direct manipulation of cell organelles or by movement of an internalized particle within the cytoplasm. However, the risk of damage that the trapping beam may induce in the biological sample due to the energy deposition has to be considered. This optically induced damage or photodamage depends mainly on the wavelength of the trapping beam, the exposure time and the biological specimen that is investigated. In this work, we explore a method to analyse the photo damage in living cells in a multimodal biophotonic workstation that is based on combining a holographic optical tweezers (HOT) microscope with a self-interference digital holographic microscopy (DHM) module. A time-dependent investigation shows that no observable changes in the cell morphology are induced at room conditions with the used laser power of the trapping beam during periods of time < 20 min of laser application. In addition, results from investigations of the photodamage increasing the working temperature to 37°C demonstrate that the optical tweezers beam can provoke severe but reversible morphology changes in the cell.

Self interference digital holographic microscopy approach for inspection of technical and biological phase specimens

Quantitative holographic phase contrast imaging enables high-resolution inspection of reflective surfaces and technical phase specimen as well as the minimally invasive analysis of living cells. However, a drawback of many experimental arrangements is the requirement for a separate reference wave which results in a phase stability decrease and the demand for a precise adjustment of the intensity ratio between object and reference wave. Thus, a self interference digital holographic microscopy (DHM) approach was explored which only requires a single object illumination wave. Due to the Michelson interferometer design of the proposed setup two wave fronts with an almost identical curvature are superimposed. This results in a nearly ideal pattern of spatial off-axis carrier fringes and a constant interferogram contrast in the hologram plane. Moreover, the hologram evaluation with spatial phase shifting reconstruction algorithms and Fourier transformation-based spatial filtering methods as well as the integration of DHM in common research microscopes is simplified. Furthermore, the use of laser light sources with a short coherence length is enabled. The applicability of the proposed self interference principle is illustrated by data from the analysis of technical and biological phase specimens. The obtained results demonstrate that the method prospects to be a versatile tool for quantitative phase contrast imaging.