Single capture bright field and off-axis digital holographic microscopy.

We report on a single capture approach for simultaneous incoherent bright field (BF) and laser-based quantitative phase imaging (QPI). Common-path digital holographic microscopy (DHM) is implemented in parallel with BF imaging within the optical path of a commercial optical microscope to achieve spatially multiplexed recording of white light images and digital off-axis holograms, which are subsequently numerically demultiplexed. The performance of the proposed multimodal concept is firstly determined by investigations on microspheres. Then, the application for label-free dual-mode QPI and BF imaging of living pancreatic tumor cells is demonstrated.

Single capture bright field and off-axis digital holographic microscopy: publisher's note.

This publisher's note contains corrections to Opt. Lett.48, 876 (2023)10.1364/OL.478674.

Refractive Index Changes of Cells and Cellular Compartments Upon Paraformaldehyde Fixation Acquired by Tomographic Phase Microscopy.

Three-dimensional quantitative phase imaging is an emerging method, which provides the 3D distribution of the refractive index (RI) and the dry mass in live and fixed cells as well as in tissues. However, an insufficiently answered question is the influence of chemical cell fixation procedures on the results of RI reconstructions. Therefore, this work is devoted to systematic investigations on the RI in cellular organelles of live and fixed cells including nucleus, nucleolus, nucleoplasm, and cytoplasm. The research was carried out on four different cell lines using a common paraformaldehyde (PFA)-based fixation protocol. The selected cell types represent the diversity of mammalian cells and therefore the results presented provide a picture of fixation caused RI changes in a broader context. A commercial Tomocube HT-1S device was used for 3D RI acquisition. The changes in the RI values after the fixation process are detected in the reconstructed phase distributions and amount to the order of 10-3 . The RI values decrease and the observed RI changes are found to be different between various cell lines; however, all of them show the most significant loss in the nucleolus. In conclusion, our study demonstrates the evident need for standardized preparation procedures in phase tomographic measurements. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

HS2ST1-dependent signaling pathways determine breast cancer cell viability, matrix interactions, and invasive behavior.

Heparan sulfate proteoglycans (HSPGs) act as signaling co-receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2-O-sulfotransferase (HS2ST1), the enzyme mediating 2-O-sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF-7 and MDA-MB-231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF-2) to HS2ST1-expressing cells compared with control cells. HS2ST1-overexpressing cells showed reduced MAPK signaling responses to FGF-2, and altered expression of epidermal growth factor receptor (EGFR), E-cadherin, Wnt-7a, and Tcf4. The increased viability of HS2ST1-depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1-dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E-cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways.

Paired Basic Amino Acid-cleaving Enzyme 4 (PCSK6): An Emerging New Target Molecule in Human Melanoma.

Although recent therapeutic developments raise hope, melanoma remains a devastating disease with a need for new treatment targets. In other tumours prohormone convertases have been shown to be pro-tumourigenic as they are involved in processing preforms of matrix-metalloproteinases, growth factors and adhesion molecules. The aim of this study was to look for new treatment options for melanoma, by investigating the role of the prohormone convertase Paired basic Amino acid-Cleaving Enzyme 4 (PACE4/PCSK6) in melanoma cell lines and human melanoma tissue. PACE4-transfected A375 melanoma cells displayed significantly increased proliferation, MMP-2 production, gelatinase activity and migratory capacity in vitro compared with sham-transfected cells. In vivo, elevated PACE4 expression resulted in significantly increased tumour growth on immunodeficient mice. In the majority of 45 human primary melanomas and melanoma metastases ex vivo PACE4 immunoreactivity was detectable, while it was absent in in situ melanomas. These results indicate PACE4 as a regulator of melanoma cell aggressiveness.

Knockdown of Musashi RNA Binding Proteins Decreases Radioresistance but Enhances Cell Motility and Invasion in Triple-Negative Breast Cancer.

The therapeutic potential of Musashi (MSI) RNA-binding proteins, important stemness-associated gene expression regulators, remains insufficiently understood in breast cancer. This study identifies the interplay between MSI protein expression, stem cell characteristics, radioresistance, cell invasiveness and migration. MSI-1, MSI-2 and Notch pathway elements were investigated via quantitative polymerase chain reaction (qPCR) in 19 triple-negative breast cancer samples. Measurements were repeated in MDA-MB-231 cells after MSI-1 and -2 siRNA-mediated double knockdown, with further experiments performed after MSI silencing. Flow cytometry helped quantify expression of CD44 and leukemia inhibitory factor receptor (LIFR), changes in apoptosis and cell cycle progression. Proliferation and irradiation-induced effects were assessed using colony formation assays. Radiation-related proteins were investigated via Western blots. Finally, cell invasion assays and digital holographic microscopy for cell migration were performed. MSI proteins showed strong correlations with Notch pathway elements. MSI knockdown resulted in reduction of stem cell marker expression, cell cycle progression and proliferation, while increasing apoptosis. Cells were radiosensitized as radioresistance-conferring proteins were downregulated. However, MSI-silencing-mediated LIFR downregulation resulted in enhanced cell invasion and migration. We conclude that, while MSI knockdown results in several therapeutically desirable consequences, enhanced invasion and migration need to be counteracted before knockdown advantages can be fully exploited.

The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased ß-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

The Influence of Capsaicin on the Integrity of Microvascular Endothelial Cell Monolayers.

Microvascular endothelial cells are an essential part of many biological barriers, such as the blood⁻brain barrier (BBB) and the endothelium of the arteries and veins. A reversible opening strategy to increase the permeability of drugs across the BBB could lead to improved therapies due to enhanced drug bioavailability. Vanilloids, such as capsaicin, are known to reversibly open tight junctions of epithelial and endothelial cells. In this study, we used several in vitro assays with the murine endothelial capillary brain cells (line cEND) as a BBB model to characterize the interaction between capsaicin and endothelial tight junctions.

Quantitative phase imaging for cell culture quality control.

The potential of quantitative phase imaging (QPI) with digital holographic microscopy (DHM) for quantification of cell culture quality was explored. Label-free QPI of detached single cells in suspension was performed by Michelson interferometer-based self-interference DHM. Two pancreatic tumor cell lines were chosen as cellular model and analyzed for refractive index, volume, and dry mass under varying culture conditions. Firstly, adequate cell numbers for reliable statistics were identified. Then, to characterize the performance and reproducibility of the method, we compared results from independently repeated measurements and quantified the cellular response to osmolality changes of the cell culture medium. Finally, it was demonstrated that the evaluation of QPI images allows the extraction of absolute cell parameters which are related to cell layer confluence states. In summary, the results show that QPI enables label-free imaging cytometry, which provides novel complementary integral biophysical data sets for sophisticated quantification of cell culture quality with minimized sample preparation. © 2017 International Society for Advancement of Cytometry.

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.

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes.

Haemolytic anaemia is one of the characteristics of life-threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34(+) haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a-mediated cellular injury of developing erythrocytes. CD34(+) haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a-binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx-mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx-producing pathogens.

Shape of red blood cells in contact with artificial surfaces.

The phenomenon of physical contact between red blood cells and artificial surfaces is considered. A fully three-dimensional mathematical model of a bilayer membrane in contact with an artificial surface is presented. Numerical results for the different geometries and adhesion intensities are found to be in agreement with experimentally observed geometries obtained by means of digital holographic microscopy.

microRNA miR-200b affects proliferation, invasiveness and stemness of endometriotic cells by targeting ZEB1, ZEB2 and KLF4.

Endometriosis is characterized by growth of endometrial tissue at ectopic locations. Down-regulation of microRNA miR-200b is observed in endometriosis and malignant disease, driving tumour cells towards an invasive state by enhancing epithelial-to-mesenchymal transition (EMT). miR-200b up-regulation may inhibit EMT and invasive growth in endometriosis. To study its functional impact on the immortalized endometriotic cell line 12Z, the stromal cell line ST-T1b, and primary endometriotic stroma cells, a transient transfection approach with microRNA precursors was employed. Expression of bioinformatically predicted targets of miR-200b was analysed by qPCR. The cellular phenotype was monitored by Matrigel invasion assays, digital-holographic video microscopy and flow cytometry. qPCR revealed significant down-regulation of ZEB1 (P < 0.05) and ZEB2 (P < 0.01) and an increase in E-cadherin (P < 0.01). miR-200b overexpression decreased invasiveness (P < 0.0001) and cell motility (P < 0.05). In contrast, cell proliferation (P < 0.0001) and the stemness-associated side population phenotype (P < 0.01) were enhanced following miR-200b transfection. These properties were possibly due to up-regulation of the pluripotency-associated transcription factor KLF4 (P < 0.05) and require attention when considering therapeutic strategies. In conclusion, up-regulation of miR-200b reverts EMT, emerging as a potential therapeutic approach to inhibit endometriotic cell motility and invasiveness.

Facing glycosphingolipid-Shiga toxin interaction: dire straits for endothelial cells of the human vasculature.

The two major Shiga toxin (Stx) types, Stx1 and Stx2, produced by enterohemorrhagic Escherichia coli (EHEC) in particular injure renal and cerebral microvascular endothelial cells after transfer from the human intestine into the circulation. Stxs are AB(5) toxins composed of an enzymatically active A subunit and the pentameric B subunit, which preferentially binds to the glycosphingolipid globotriaosylceramide (Gb3Cer/CD77). This review summarizes the current knowledge on Stx-caused cellular injury and the structural diversity of Stx receptors as well as the initial molecular interaction of Stxs with the human endothelium of different vascular beds. The varying lipoforms of Stx receptors and their spatial organization in lipid rafts suggest a central role in different modes of receptor-mediated endocytosis and intracellular destiny of the toxins. The design and development of tailored Stx neutralizers targeting the oligosaccharide-toxin recognition event has become a very real prospect to ameliorate or prevent life-threatening renal and neurological complications.

Quantitative Phase Imaging as Sensitive Screening Method for Nanoparticle-Induced Cytotoxicity Assessment.

The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC50) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.

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.

Application of Digital Holographic Microscopy to Analyze Changes in T-Cell Morphology in Response to Bacterial Challenge.

Quantitative phase imaging (QPI) is a non-invasive, label-free technique used to detect aberrant cell morphologies caused by disease, thus providing a useful diagnostic approach. Here, we evaluated the potential of QPI to differentiate specific morphological changes in human primary T-cells exposed to various bacterial species and strains. Cells were challenged with sterile bacterial determinants, i.e., membrane vesicles or culture supernatants, derived from different Gram-positive and Gram-negative bacteria. Timelapse QPI by digital holographic microscopy (DHM) was applied to capture changes in T-cell morphology over time. After numerical reconstruction and image segmentation, we calculated single cell area, circularity and mean phase contrast. Upon bacterial challenge, T-cells underwent rapid morphological changes such as cell shrinkage, alterations of mean phase contrast and loss of cell integrity. Time course and intensity of this response varied between both different species and strains. The strongest effect was observed for treatment with S. aureus-derived culture supernatants that led to complete lysis of the cells. Furthermore, cell shrinkage and loss of circular shape was stronger in Gram-negative than in Gram-positive bacteria. Additionally, T-cell response to bacterial virulence factors was concentration-dependent, as decreases in cellular area and circularity were enhanced with increasing concentrations of bacterial determinants. Our findings clearly indicate that T-cell response to bacterial stress depends on the causative pathogen, and specific morphological alterations can be detected using DHM.

Quantitative Phase Imaging Using Digital Holographic Microscopy to Assess the Degree of Intestinal Inflammation in Patients with Ulcerative Colitis.

Ulcerative colitis (UC) is characterized by chronic inflammation of the colorectum. Histological remission has emerged as a potential future treatment goal; however, the histopathological assessment of intestinal inflammation in UC remains challenging with a multitude of available scoring systems and the need for a pathologist with expertise in inflammatory bowel disease (IBD). In previous studies, quantitative phase imaging (QPI) including digital holographic microscopy (DHM) was successfully applied as an objective method for stain-free quantification of the degree of inflammation in tissue sections. Here, we evaluated the application of DHM for the quantitative assessment of histopathological inflammation in patients with UC. In our study, endoscopically obtained colonic and rectal mucosal biopsy samples from 21 patients with UC were analyzed by capturing DHM-based QPI images that were subsequently evaluated using the subepithelial refractive index (RI). The retrieved RI data were correlated with established histological scoring systems including the Nancy index (NI) as well as with endoscopic and clinical findings. As a primary endpoint, we found a significant correlation between the DHM-based retrieved RI and the NI (R2 = 0.251, p < 0.001). Furthermore, RI values correlated with the Mayo endoscopic subscore (MES; R2 = 0.176, p < 0.001). An area under the receiver operating characteristics (ROC) curve of 0.820 confirms the subepithelial RI as a reliable parameter to distinguish biopsies with histologically active UC from biopsies without evidence of active disease as determined by conventional histopathological examination. An RI higher than 1.3488 was found to be the most sensitive and specific cut-off value to identify histologically active UC (sensitivity of 84% and specificity of 72%). In conclusion, our data demonstrate DHM to be a reliable tool for the quantitative assessment of mucosal inflammation in patients with UC.

Durable 3D murine ex vivo retina glaucoma models for optical coherence tomography.

Durable and standardized phantoms with optical properties similar to native healthy and disease-like biological tissues are essential tools for the development, performance testing, calibration and comparison of label-free high-resolution optical coherence tomography (HR-OCT) systems. Available phantoms are based on artificial materials and reflect thus only partially ocular properties. To address this limitation, we have performed investigations on the establishment of durable tissue phantoms from ex vivo mouse retina for enhanced reproduction of in vivo structure and complexity. In a proof-of-concept study, we explored the establishment of durable 3D models from dissected mouse eyes that reproduce the properties of normal retina structures and tissue with glaucoma-like layer thickness alterations. We explored different sectioning and preparation procedures for embedding normal and N-methyl-D-aspartate (NMDA)-treated mouse retina in transparent gel matrices and epoxy resins, to generate durable three-dimensional tissue models. Sample quality and reproducibility were quantified by thickness determination of the generated layered structures utilizing computer-assisted segmentation of OCT B-scans that were acquired with a commercial HR-OCT system at a central wavelength of 905 nm and analyzed with custom build software. Our results show that the generated 3D models feature thin biological layers close to current OCT resolution limits and glaucoma-like tissue alterations that are suitable for reliable HR-OCT performance characterization. The comparison of data from resin-embedded tissue with native murine retina in gels demonstrates that by utilization of appropriate preparation protocols, highly stable samples with layered structures equivalent to native tissues can be fabricated. The experimental data demonstrate our concept as a promising approach toward the fabrication of durable biological 3D models suitable for high-resolution OCT system performance characterization supporting the development of optimized instruments for ophthalmology applications.

Dysregulated Stem Cell Markers Musashi-1 and Musashi-2 are Associated with Therapy Resistance in Inflammatory Breast Cancer.

BACKGROUND AND AIM: While preliminary evidence points to pro-tumorigenic roles for the Musashi (MSI) RNA-binding proteins Musashi-1 (MSI1) and Musashi-2 (MSI2) in some breast cancer subtypes, no data exist for inflammatory breast cancer (IBC). METHODS: MSI gene expression was quantified in IBC SUM149PT cells. We then used small interfering RNA-based MSI1 and MSI2 double knockdown (DKD) to understand gene expression and functional changes upon MSI depletion. We characterized cancer stem cell characteristics, cell apoptosis and cell cycle progression via flow cytometry, mammospheres via spheroid assays, migration and proliferation via digital holographic microscopy, and cell viability using BrdU assays. Chemoresistance was determined for paclitaxel and cisplatin with MTT assays and radioresistance was assessed with clonogenic analyses. In parallel, we supported our in vitro data by analyzing publicly available patient IBC gene expression datasets. RESULTS: MSI1 and MSI2 are upregulated in breast cancer generally and IBC specifically. MSI2 is more commonly expressed compared to MSI1. MSI DKD attenuated proliferation, cell cycle progression, migration, and cell viability while increasing apoptosis. Stem cell characteristics CD44(+)/CD24(-), TERT and Oct4 were associated with MSI expression in vivo and were decreased in vitro after MSI DKD as was ALDH expression and mammosphere formation. In vivo, chemoresistant tumors were characterized by MSI upregulation upon chemotherapy application. In vitro, MSI DKD was able to alleviate chemo- and radioresistance. CONCLUSIONS: The Musashi RNA binding proteins are dysregulated in IBC and associated with tumor proliferation, cancer stem cell phenotype, chemo- and radioresistance. MSI downregulation alleviates therapy resistance and attenuates tumor proliferation in vitro.