In holographic tomography obtaining projections is the key part of the measurement process that enables the 3D refractive index reconstruction. The error of the illumination angle can significantly influence the reconstruction quality and alter the result degrading the reliability of the reconstruction. Thus, in this paper the impact of the scanning errors in limited angle holographic tomography with respect to two reconstruction algorithms is analyzed. The simulated errors are compared to the errors identified in the experimental system. The reconstruction errors are verified using a paramecium cell phantom at the simulation stage and with a biological object, namely a macrophage cell in the experimental part. The experimental system presented in the paper exhibits maximum expected measurement errors found in galvanometer-mirror-based holographic tomography setups.
During long-term imaging, cells move out of the field of view. We have generated functionalized substrates containing rectangular areas, which were capable in keeping cells over the whole observation period.
Digital holographic microscopy (DHM) was used as multimodal optical method for nanomaterial toxicity testing that overcomes the limitations and assay disturbances of conventional in vitro assays based on absorbance or fluorescence read outs.
The major problem of Digital Holographic Microscopy (DHM) long term live cell imaging is that over time most of the tracked cells move out of the image area and other ones move in. Therefore, most of the cells are lost for the evaluation of individual cellular processes. Here, we present an effective solution for this crucial problem of long-term microscopic live cell analysis. We have generated functionalized slides containing areas of 250 μm per 200 μm. These micropatterned biointerfaces consist of passivating polyaclrylamide brushes (PAAm). Inner areas are backfilled with octadecanthiol (ODT), which allows cell attachment. The fouling properties of these surfaces are highly controllable and therefore the defined areas designed for the size our microscopic image areas were effective in keeping all cells inside the rectangles over the selected imaging period.
The cytotoxic potential of nanomaterials is commonly evaluated by different cellular endpoints as reactive oxygen species formation, cell viability or cell death. Usually these parameters are determined by intensity based optical readouts that are often influenced by nanomaterial-based interferences. Here we present Digital Holographic Microscopy (DHM) as a multimodal optical method, which overcomes the limitations of conventional in vitro assays based on color or fluorescence read outs. Using cell viability WST8- and cell death LDH-assay we investigated the toxic effects of two representative silver nanomaterials. Therefore, we used a matrix of four cell lines representing different organ functions. Compared to conventional toxicity assays DHM allows time resolved proliferation monitoring which is free of assay system interactions. Also, information about time-dependent mechanisms can be obtained. Additionally, we have analyzed single macrophages for refractive index, cell volume and dry mass after the incubation to cytotoxic silver spheres. The refractive index decreased dose dependent, while cell volume and dry mass stayed constant. We therefore suggest the evaluation of these parameters in cytotoxicity assessment for further evaluation of their relevance under the applied conditions. This demonstrates DHM as valuable label-free tool for nanomaterial toxicity analysis.
Commonly used cytotoxicity assays to determine the formation of reactive oxygen species, cell viability or cell death are often affected by applied nanomaterials, which lead to false-positive or false-negative results. Thus, novel nanomaterial toxicity testing strategies that allow for high nanomaterial doses to determine Low Effect Levels (LOEL) even of low toxic materials are of high interest. We demonstrate novel approaches to quantify cytotoxic effects with new parameter sets such as cellular refractive index, volume, density and dry mass that are obtained by digital holographic microscopy (DHM). Furthermore, we correlate results obtained from spherical (NM 300) and rod shaped (NM 302) silver nanomaterials with established cell viability and cell death assays. Moreover, in a label-free flow cytometry configuration, cell-nanoparticle-interaction-kinetics were determined by side scatter signal analysis. We demonstrate that silver spheres show a higher cytotoxicity than silver rods and found that this effect correlates with a decrease of the intracellular refractive index and a decreased temporal development of dry mass and cell covered surface area indicating reduced cell viability and increased cell death. Results from side scatter analysis suggest a dose-dependent uptake kinetics of both materials that correlates with cytotoxicity data of the established assays. Taken together, our results demonstrate DHM and flow cytometry as promising novel label-free tools for nanomaterial toxicity and cell particle interaction studies.
The in vitro cytotoxicity assessment of engineered nanoparticles commonly involves the measurement of different endpoints like the formation of reactive oxygen species, cell viability or cell death. Usually these parameters are determined by optical readouts of enzymatically converted substrates that often interfere with the tested nanomaterials. Using cell viability (WST-8) and cell death (LDH) as parameter we have initially investigated the toxic effects of spherical (NM 300) and rod shaped (NM 302) silver nanomaterials with a matrix of four cell lines representing different functions: lung and kidney epithelial cells, macrophages and fibroblasts. In addition, we have used a label-free flow cytometer configuration to investigate interactions of particles and macrophages by side scatter signal analysis. Finally, we explored digital holographic microscopy (DHM) for multimodal label-free analysis of nanomaterial toxicity. Quantitative DHM phase images were analyzed for cell thickness, volume, density, dry mass and refractive index. We could demonstrate that silver spheres lead to more cytotoxic effects than rods in all four examined cell lines and both assay. Exemplarily a dose dependent interaction increase of cells with NM 300 and NM 302 analyzed by flow cytometry is shown. Furthermore, we found that the refractive index of cells is influenced by incubation with NM 300 in a decreasing manner. A 24 hours time-lapse measurement revealed a dose dependent decrease of dry mass and surface area development indicating reduced cell viability and cell death. Our results demonstrate digital holographic microscopy and flow cytometry as valuable label-free tools for nanomaterial toxicity and cell interaction studies.
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