Angelika Vollmer

Simplified approach for quantitative digital holographic phase contrast imaging of living cells

Many interferometry-based quantitative phase contrast imaging techniques require a separately generated coherent reference wave. This results in a low phase stability and the demand for a precise adjustment of the intensity ratio between object and reference wave. To overcome these problems, the performance of a Michelson interferometer approach for digital holographic microscopy was analyzed that avoids a separately generated reference wave by superposition of different image areas. It is shown that this simplified arrangement yields improved phase stability. Furthermore, results from time-lapse investigations on living pancreas tumor cells demonstrate the capability of the method for reliable quantitative phase contrast imaging.

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.

Tomographic phase microscopy of living three-dimensional cell cultures

Abstract. A successful application of self-interference digital holographic microscopy in combination with a sample-rotation-based tomography module for three-dimensional (3-D) label-free quantitative live cell imaging with subcellular resolution is demonstrated. By means of implementation of a hollow optical fiber as the sample cuvette, the observation of living cells in different 3-D matrices is enabled. The fiber delivers a stable and accurate rotation of a cell or cell cluster, providing quantitative phase data for tomographic reconstruction of the 3-D refractive index distribution with an isotropic spatial resolution. We demonstrate that it is possible to clearly distinguish and quantitatively analyze several cells grouped in a “3-D cluster” as well as subcellular organelles like the nucleoli and local internal refractive index changes.

Three-Dimensional Exploration and Mechano-Biophysical Analysis of the Inner Structure of Living Cells

A novel mechanobiological method is presented to explore qualitatively and quantitatively the inside of living biological cells in three dimensions, paving the way to sense intracellular changes during dynamic cellular processes. For this purpose, holographic optical tweezers, which allow the versatile manipulation of nanoscopic and microscopic particles by means of tailored light fields, are combined with self-interference digital holographic microscopy. This biophotonic holographic workstation enables non-contact, minimally invasive, flexible, high-precision optical manipulation and accurate 3D tracking of probe particles that are incorporated by phagocytosis in cells, while simultaneously quantitatively phase imaging the cell morphology. In a first model experiment, internalized polystyrene microspheres with 1 μm diameter are three-dimensionally moved and tracked in order to quantify distances within the intracellular volume with submicrometer accuracy. Results from investigations on cell swelling provoked by osmotic stimulation demonstrate the homogeneous stretching of the cytoskeleton network, and thus that the proposed method provides a new way for the quantitative 3D analysis of the dynamic intracellular morphology.

Towards 3D modelling and imaging of infection scenarios at the single cell level using holographic optical tweezers and digital holographic microscopy.

The analysis of dynamic interactions of microorganisms with a host cell is of utmost importance for understanding infection processes. We present a biophotonic holographic workstation that allows optical manipulation of bacteria by holographic optical tweezers and simultaneously monitoring of dynamic processes with quantitative multi-focus phase imaging based on self-interference digital holographic microscopy. Our results show that several bacterial cells, even with non-spherical shape, can be aligned precisely on the surface of living host cells and localized reproducibly in three dimensions. In this way a new label-free multipurpose device for modelling and quantitative analysis of infection scenarios at the single cell level is provided.

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.

Application of Color Digital Holographic Microscopy for Analysis of Stained Tissue Sections

The analysis of stained tissue sections represents an important tool in medical diagnostics. Color digital holographic microscopy offers subsequent multi-focus true color imaging with simultaneous quantitative phase contrast analysis. Investigations on digital recording and numerical reconstructions of color digital holographic images have been performed by applying a transmission microscope experimental setup in holographic off-axis geometry. Three monochromatic digital holograms with different wavelengths in red, green and blue spectral range are recorded. After digital holographic refocusing and compensation for aberrations of the microscope imaging system by digital image correlation the numerically reconstructed amplitude distributions are combined into color images. The applicability of the method is demonstrated by results obtained from stained intestine tissue sections.

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.