Digital holographic microscopy (DHM) has been demonstrated as a label-free minimally-invasive quantitative phase imaging (QPI) tool to assess the cytotoxic potential of nanoparticles in vitro. Due to low laser light intensities DHM minimizes interactions with the sample. The analyzed cell cultures thus remain in a native state, making them available for further downstream analysis. In a study on mouse macrophages (RAW 264.7) we explored if cell populations can be analyzed with common biochemical assays after the DHM-based QPI observation. Therefore, the mean effective concentration (EC50) of poly(alkyl cyanoacrylate) nanocarriers was firstly determined based on the DHM accessible dry mass increment after 24 h. The results were then compared to corresponding data from two subsequently performed colorimetric cell viability and cytotoxicity assays. EC50 values from all assays are found in good agreement and demonstrate the potential of QPI to achieve an increased data quality in in vitro cytotoxicity experiments.
Digital holographic microscopy (DHM) has been demonstrated to be a suitable label-free and non-invasive quantitative phase imaging (QPI) tool in risk assessment of the cytotoxic potential of engineered nanoparticles and organic nanocarriers. For a broader application, robustness of DHM-based assays needs to be demonstrated towards DHM standardization in risk assessment. Thus, we performed an interlaboratory comparison on the transferability and reproducibility of a DHM-based assay. The cytotoxic potential of organic nanoparticles on A549 lung epithelial cells was analyzed in two European laboratories using identically constructed DHM systems. Our results demonstrate a solid and accurate performance of the DHM-based cytotoxicity assay
Nanoparticles are currently the subject of numerous research activities due to their unique physical and chemical properties, such as for industrial applications, but also in studies on environmental toxicity and human health effects. Since the toxicity of nanoparticles is mainly based on their interaction and uptake by cells, the aim of this study was to quantify their cellular internalization. Quantitative phase imaging (QPI) has proven to be a versatile method for minimally invasive label-free imaging of biological specimens, whereas laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) allows the label-free identification and quantification of nanomaterials in biological tissues. However, the determination of nanoparticle concentrations within single cells in vitro is challenging for LA-ICP-MS as precise information about the cell volume is required. We thus combined the LA-ICP-MS analysis with digital holographic microscopy (DHM), a QPI technology, which allows single cell volume determination prior to measurements by LA-ICP-MS. The implementation of this method at the single cell level in vitro was investigated by determining the concentration of Ce in RLE-6TN lung epithelial cells after exposition to CeO2 nanomaterial. Results from correlative LA-ICP-MS and DHM QPI investigations on nanoparticle-loaded cells show significant effects on intracellular Ce-levels for RLE-6TN cells treated with different CeO2 nanoparticle concentrations and therefore demonstrate the feasibility of this new concept.
Significant improvements in quantitative phase imaging (QPI) technologies established digital holographic microscopy (DHM) and holotomography (HT) as tools for label-free imaging of cell morphology as well as to assess the 3D distribution of intracellular structures. Here, we applied DHM and HT consecutively on the same samples to observe and quantify the impact of nanomaterials on the morphology of primary hepatocytes. The liver has a high vascularization and metabolic activity, and hepatocytes were selected because they may contact and internalize nanoparticles circulating in the blood. Effects of nanoparticles on cells can range from a reduction of viability to alterations in morphology and intracellular structures. Thus, first an automated modular DHM setup was applied for large-area QPI screening of the entire hepatocyte populations while a commercial HT system (Tomocube HT-2H, Tomocube, Korea) was utilized to observe selected tiny 3-dimensional intracellular changes of interest via refractive index tomograms. For the investigations, hepatocytes were isolated from collagenase-perfused rat livers and seeded into HT-compatible cell culture dishes. After cultivation and incubation with different types of nanoparticles (CeO2, Ag, Au) for 24 hours the cells were fixed with a mixture of glutaraldehyde and paraformaldehyde to preserve cell morphology and structure. The results of our study demonstrate that QPI with DHM is capable for efficient large-area 2D screening and to reveal of nanomaterial-related alterations in the entire hepatocyte populations while HT provides high performance complementary 3D insights and the localization of tiny intracellular damages.
In laser based digital holographic microscopy (DHM) quantitative phase imaging (QPI) is affected by scattering and interference fringes due to internal reflections in the experimental setup. We present a concept for the reduction of such coherence induced disturbances. In our approach, the sample illumination light is modulated by an electrically focus tunable lens while series of digital off-axis holograms are recorded from which subsequently averaged QPI images are calculated. The concept is compatible with Mach-Zehnder interferometer-based off-axis DHM and capable for usage with commercial research microscopes. The performance is illustrated by results from living cells.
In digital holographic microscopy (DHM) scattering patterns and parasitic interference fringes due to internal reflections that are caused by coherent laser light affect quantitative phase imaging (QPI). We present an approach for the reduction coherence induced disturbances. Therefore, the sample illumination is modulated by an electrically focus tunable lens while a series of digital off-axis holograms is recorded from which subsequently averaged QPI images are retrieved. The principle is compatible with Mach-Zehnder interferometer-based off-axis DHM and capable to be integrated into commercial research microscopes. The performance of our approach is demonstrated by results from living porcine epithelia cells and mouse fibroblasts.
Quantitative phase imaging (QPI) has been demonstrated to be a versatile tool for minimally invasive label-free imaging of biological specimens and time-resolved cellular analysis. In cytotoxicity testing, a commonly performed task in the preclinical development of new medical products, a flexible time-resolved label-free quantification of drug mediated effects is highly desirable. We thus explored the capabilities of digital holographic microscopy (DHM), an interferometric variant of QPI, to determine the temporal dry mass development of a cell population of RAW 264.7 mouse macrophages to quantify effects caused by medical nanoparticles in vitro in comparison to digitonin, which was used as cytotoxic positive control, and untreated cells in culture medium. Digital off-axis holograms from medical nanoparticles and digitonin incubated cells as well as from untreated control cells were acquired automated in multi-well imaging plates using a modular DHM system that was equipped with a motorized stage. From the retrieved time-lapse series of quantitative phase images, the temporal development of the dry mass in the observed field of view was calculated. As dry mass is correlated to cellular proliferation, toxicological effects of the medical nanoparticles on macrophages can be quantified at the nanogram scale. We found a similar temporal dry mass development of control macrophages in the cell culture medium and after incubation with non-toxic liposomes, while cytotoxic poly(alkyl cyanoacrylate) nanoparticles significantly reduced the dry mass increment in a dose-dependent manner. In summary, our results show for macrophages that the observation of the temporal dry mass development allows to discriminate reliably between non(low)-toxic and harmful nanomaterials.
We explored a Mask Region-based Convolutional Neural Network (Mask R-CNN) to detect macrophages in quantitative phase images, which were acquired by digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI). The Mask R-CNN deep learning architecture is capable to detect and segment single macrophage cells in quantitative phase images and allows to perform both tasks in a multi-stage process. Our results show that the combined detection and segmentation of cells through Mask R-CNN-based automated evaluation prospects a fast and robust screening in label-free high throughput microscopy.
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