Grading of glioma tumors using digital holographic microscopy.

Gliomas are the most common type of cerebral tumors; they occur with increasing incidence in the last decade and have a high rate of mortality. For efficient treatment, fast accurate diagnostic and grading of tumors are imperative. Presently, the grading of tumors is established by histopathological evaluation, which is a time-consuming procedure and relies on the pathologists' experience. Here we propose a supervised machine learning procedure for tumor grading which uses quantitative phase images of unstained tissue samples acquired by digital holographic microscopy. The algorithm is using an extensive set of statistical and texture parameters computed from these images. The procedure has been able to classify six classes of images (normal tissue and five glioma subtypes) and to distinguish between gliomas types from grades II to IV (with the highest sensitivity and specificity for grade II astrocytoma and grade III oligodendroglioma and very good scores in recognizing grade III anaplastic astrocytoma and grade IV glioblastoma). The procedure bolsters clinical diagnostic accuracy, offering a swift and reliable means of tumor characterization and grading, ultimately the enhancing treatment decision-making process.

Early differentiation of mesenchymal stem cells is reflected in their dielectrophoretic behavior.

The therapeutic use of mesenchymal stem cells (MSCs) becomes more and more important due to their potential for cell replacement procedures as well as due to their immunomodulatory properties. However, protocols for MSCs differentiation can be lengthy and may result in incomplete or asynchronous differentiation. To ensure homogeneous populations for therapeutic purposes, it is crucial to develop protocols for separation of the different cell types after differentiation. In this article we show that, when MSCs start to differentiate towards adipogenic or osteogenic progenies, their dielectrophoretic behavior changes. The values of cell electric parameters which can be obtained by dielectrophoretic measurements (membrane permittivity, conductivity, and cytoplasm conductivity) change before the morphological features of differentiation become microscopically visible. We further demonstrate, by simulation, that these electric modifications make possible to separate cells in their early stages of differentiation by using the dielectrophoretic separation technique. A label free method which allows obtaining cultures of homogenously differentiated cells is thus offered.

OpenDEP: An Open-Source Platform for Dielectrophoresis Spectra Acquisition and Analysis.

Dielectrophoretic (DEP) cell separation, which utilizes electric fields to selectively manipulate and separate cells based on their electrical properties, has emerged as a cutting-edge label-free technique. DEP separation techniques rely on differences in the electrical and morphological properties of cells, which can be obtained by a thorough analysis of DEP spectra. This article presents a novel platform, named OpenDEP, for acquiring and processing DEP spectra of suspended cells. The platform consists of lab-on-a-chip and open-source software that enables the determination of DEP spectra and electric parameters. The performance of OpenDEP was validated by comparing the results obtained using this platform with the results obtained using a commercially available device, 3DEP from DEPtech. The lab-on-a-chip design features two indium tin oxide-coated slides with a specific geometry, forming a chamber where cells are exposed to an inhomogeneous alternating electric field with different frequencies, and microscopic images of cell distributions are acquired. A custom-built software written in the Python programing language was developed to convert the acquired images into DEP spectra, allowing for the estimation of membrane and cytoplasm conductivities and permittivities. The platform was validated using two cell lines, DC3F and NIH 3T3. The OpenDEP platform offers several advantages, including easy manufacturing, statistically robust computations due to large cell population analysis, and a closed environment for sterile work. Furthermore, continuous observation using any microscope allows for integration with other techniques.

Method for nanoparticles uptake evaluation based on double labeled fluorescent cells scanned in enhanced darkfield microscopy.

We present a method that integrates the standard imaging tools for locating and detecting unlabeled nanoparticles (NPs) with computational tools for partitioning cell volumes and NPs counting within specified regions to evaluate their internal traffic. The method uses enhanced dark field CytoViva optical system and combines 3D reconstructions of double fluorescently labeled cells with hyperspectral images. The method allows the partitioning of each cell image into four regions: nucleus, cytoplasm, and two neighboring shells, as well as investigations across thin layers adjacent to the plasma membrane. MATLAB scripts were developed to process the images and to localize NPs in each region. Specific parameters were computed to assess the uptake efficiency: regional densities of NPs, flow densities, relative accumulation indices, and uptake ratios. The results of the method are in line with biochemical analyses. It was shown that a sort of saturation limit for intracellular NPs density is reached at high extracellular NPs concentrations. Higher NPs densities were found in the proximity of the plasma membranes. A decrease of the cell viability with increasing extracellular NPs concentration was observed and explained the negative correlation of the cell eccentricity with NPs number.

Evaluation of intracellular distribution of folate functionalized silica nanoparticles using fluorescence and hyperspectral enhanced dark field microscopy.

Using nanoparticles as carriers for drug delivery systems has become a widely applied strategy in therapeutics and diagnostics. However, the pattern of their intracellular distribution is yet to be clarified. Here we present an in vitro study on the incorporation of mesoporous silica nanoparticles conjugated with folate and loaded with a cytotoxic drug, Irinotecan. The nanoparticles count and distribution within the cell frame were evaluated by means of enhanced dark field microscopy combined with hyperspectral imagery and 3D reconstructions from double-labeled fluorescent samples. An original post-processing procedure was developed to emphasize the nanoparticles' localization in 3D reconstruction of cellular compartments. By these means, it has been shown that the conjugation of mesoporous silica nanoparticles with folate increases the efficiency of nanoparticles entering the cell and their preferential localization in the close vicinity of the nucleus. As revealed by metabolic viability assays, the nanoparticles functionalized with folate enhance the cytotoxic efficiency of Irinotecan.

Facile synthesis of low toxicity iron oxide/TiO2 nanocomposites with hyperthermic and photo-oxidation properties.

The present study aimed to assess the feasibility of developing low-cost multipurpose iron oxide/TiO2 nanocomposites (NCs) for use in combined antitumor therapies and water treatment applications. Larger size (≈ 100 nm) iron oxide nanoparticles (IONPs) formed magnetic core-TiO2 shell structures at high Fe/Ti ratios and solid dispersions of IONPs embedded in TiO2 matrices when the Fe/Ti ratio was low. When the size of the iron phase was comparable to the size of the crystallized TiO2 nanoparticles (≈ 10 nm), the obtained nanocomposites consisted of randomly mixed aggregates of TiO2 and IONPs. The best inductive heating and ROS photogeneration properties were shown by the NCs synthesized at 400 °C which contained the minimum amount of α-Fe2O3 and sufficiently crystallized anatase TiO2. Their cytocompatibility was assessed on cultured human and murine fibroblast cells and analyzed in relation to the adsorption of bovine serum albumin from the culture medium onto their surface. The tested nanocomposites showed excellent cytocompatibility to human fibroblast cells. The results also indicated that the environment (i.e. phosphate buffer or culture medium) used to disperse the nanomaterials prior to performing the viability tests can have a significant impact on their cytotoxicity.

Digital holographic microscopy evaluation of dynamic cell response to electroporation.

Phase-derived parameters and time autocorrelation functions were used to analyze the behavior of murine B16 cells exposed to different amplitudes of electroporation pulses. Cells were observed using an off-axis digital holographic microscope equipped with a fast camera. Series of quantitative phase images of cells were reconstructed and further processed using MATLAB codes. Projected area, dry mass density, and entropy proved to be predictors for permeabilized cells that swell or collapse. Autocorrelation functions of phase fluctuations in different regions of the cell showed a good correlation with the local effectiveness of permeabilization.

Changes in the packing of bilayer lipids triggered by electroporation: real-time measurements on cells in suspension.

Electropermeabilization of the cell membrane is a technique used to facilitate penetration of impermeant molecules into cells. Although there are studies regarding the mechanism of processes occurring after electropermeabilization, the relationship between electropermeabilization and associated phenomena (e.g. generation of reactive oxygen species, endocytosis, lipid peroxidation, etc.) is yet to be elucidated. This work aimed to get information on the changes in the packing of the bilayer lipids and their peroxidation induced by application of electroporation pulses. We used a specially designed system of electrodes which allowed performing electropermeabilization of cells in suspension simultaneously with time-dependent measurements of fluorescence and temperature. The kinetics of membrane packing and production of reactive oxygen species were studied using various conductivity buffers (0.01, 0.04 and 0.14 S/m) and different number of 1 kV/cm bipolar pulses (1-50). Two categories of effects were observed: a thermal effect, consisting in an increased bilayer disorder (a deeper penetration of water into the hydrophobic core), and a nonthermal effect, leading to a higher degree of lipids packing, the latter being attributed to a peroxidation process. An analysis of the permeabilization conditions in which one of these two processes predominates was performed.

Influence of surfactant-tailored Mn-doped ZnO nanoparticles on ROS production and DNA damage induced in murine fibroblast cells.

The present study concerns the in vitro oxidative stress responses of non-malignant murine cells exposed to surfactant-tailored ZnO nanoparticles (NPs) with distinct morphologies and different levels of manganese doping. Two series of Mn-doped ZnO NPs were obtained by coprecipitation synthesis method, in the presence of either polyvinylpyrrolidone (PVP) or sodium hexametaphosphate (SHMTP). The samples were investigated by powder X-ray Diffraction, Transmission Electron Microscopy, Fourier-Transform Infrared and Electron Paramagnetic Resonance spectroscopic methods, and N2 adsorption-desorption analysis. The observed surfactant-dependent effects concerned: i) particle size and morphology; ii) Mn-doping level; iii) specific surface area and porosity. The relationship between the surfactant dependent characteristics of the Mn-doped ZnO NPs and their in vitro toxicity was assessed by studying the cell viability, intracellular reactive oxygen species (ROS) generation, and DNA fragmentation in NIH3T3 fibroblast cells. The results indicated a positive correlation between the specific surface area and the magnitude of the induced toxicological effects and suggested that Mn-doping exerted a protective effect on cells by diminishing the pro-oxidative action associated with the increase in the specific BET area. The obtained results support the possibility to modulate the in vitro toxicity of ZnO nanomaterials by surfactant-controlled Mn-doping.

Effects of Ibrutinib on biophysical parameters of platelet in patients with chronic lymphocytic leukaemia.

Patients with chronic lymphocytic leukemia (CLL) treated with Ibrutinib often present hemorrhagic complications. Platelets dysfunction is well documented by aggregometry and flow cytometry, but the mechanisms by which Ibrutinib treatment influences the platelets status is yet to be evaluated. The aim of this study is to identify platelet membrane parameters in chronic lymphocytic leukemia (CLL) that could be altered by Ibrutinib administration. In this paper we propose a set of fluorescence measurements of the following parameters: membrane fluidity, resting membrane potential, and reactive oxygen species production of platelets suspensions obtained from CLL patients treated or not with Ibrutinib as markers for platelets status in this pathological situation. Platelets from CLL patients treated with Ibrutinib have higher membrane fluidity, lower resting membrane potential and higher level of reactive oxygen species production compared to the untreated CLL patients. These patients are also presenting higher membrane fluidity and lower resting membrane potential compared to healthy volunteers.

An experimental system for real-time fluorescence recordings of cell membrane changes induced by electroporation.

The electroporation of cells is nowadays used for a large variety of purposes, from basic research to cancer therapy and food processing. Understanding molecular mechanisms of the main processes involved in electroporation is thus of significant interest. In the present work, we propose an experimental system to record in real time the evolution of any cell parameter which can be evaluated by fluorescence (before, during and after application of the electroporation pulses to cells in suspension). The system is based on the design of adequate electroporation electrodes, compatible with a standard spectrofluorometer cuvette housing. The electric field intensity generated when pulses are applied was carefully characterized for different geometries of the electrodes, to choose a construction ensuring the greatest homogeneity of the field in combination with the best possible illumination of the sample. As an example of the method's application, we present here generalized polarization kinetics for a varying number of electroporation pulses applied to a cell suspension; the general polarization parameter is strongly correlated to water presence in the hydrophobic membrane core. The system may be used for many other fluorescence measurements useful for the characterization of the electroporation process.

Noninvasive detection of changes in cells' cytosol conductivity by combining dielectrophoresis with optical tweezers.

Cellular electrical properties are modulated by various physical and/or chemical stresses and detection of these changes is a challenging issue. Optical tweezers (OT) and dielectrophoresis (DEP) are frequently integrated to devices dedicated to the investigation of cells properties. Here we provide a technique to detect changes in cytosol conductivity of cells by using a combination of DEP and OT. The method was exemplified for the case of cells electroporation and is based on balancing the DEP force by a controlled OT force. We observed a decrease of the DEP force in the case of electroporated cells which was correlated to a decrease of cytosol conductivity by means of Clausius-Mossotti factor modeling. For highly stressing electroporation pulses, the cytosol conductivity drops to values close to those of the cells suspending medium. These results are consistent with those reported in the literature proving the robustness of our proposed sensing method.

Evaluation of the metastatic potential of malignant cells by image processing of digital holographic microscopy data.

The cell refractive index has been proposed as a putative cancer biomarker of great potential, being correlated with cell content and morphology, cell division rate and membrane permeability. We used digital holographic microscopy to compare the refractive index and dry mass density of two B16 murine melanoma sublines of different metastatic potential. Using statistical methods, the distribution of phase shifts within the reconstructed quantitative phase images was analyzed by the method of bimodality coefficients. The observed correlation of refractive index, dry mass density and bimodality profile with the metastatic potential of the cells was validated by real time impedance-based assay and clonogenic tests. We suggest that the refractive index and bimodality analysis of quantitative phase image histograms could be developed as optical biomarkers useful in label-free detection and quantitative evaluation of cell metastatic potential.

Changes in optical properties of electroporated cells as revealed by digital holographic microscopy.

Changes in optical and shape-related characteristics of B16F10 cells after electroporation were investigated using digital holographic microscopy (DHM). Bipolar rectangular pulses specific for electrochemotherapy were used. Electroporation was performed in an "off-axis" DHM set-up without using exogenous markers. Two types of cell parameters were monitored seconds and minutes after pulse train application: parameters addressing a specifically defined area of the cell (refractive index and cell height) and global cell parameters (projected area, optical phase shift profile and dry mass). The biphasic behavior of cellular parameters was explained by water and mannitol dynamics through the electropermeabilized cell membrane.

Hemorrhagic risk due to platelet dysfunction in myelodysplastic patients, correlations with anemia severity and iron overload.

Platelet function is influenced by changes in membrane fluidity that has an important role in the expression of platelet receptors and in modulating the activity of proteins like phospholipase C or proteinkinase C. In freshly prepared platelets, membrane fluidity modifies the aggregation/agglutination function. Reactive oxygen species (ROS) represent another important parameter involved in platelet receptor activation. There is a certain association of high levels of ROS and iron overload. Patients with hemochromatosis have low platelet aggregation induced by thrombin; little is known about the anemia and effects of iron overload on platelet activation in myelodysplastic syndromes (MDS) patients. Study of platelet membrane fluidity and ROS production changes in patients with MDS and possible correlations with altered platelet function as reflected in aggregation curves and platelet receptor expression. To find out possible correlations of fluidity of platelet membrane and ROS level with hematologic parameters and iron levels. The prospective study included 34 patients with myelodysplastic syndromes classified according to French-American-British cooperative group proposals and 29 healthy volunteers. Platelet membrane fluidity was quantified by fluorescence anisotropy measurements using the marker 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. ROS production was evaluated by fluorescence measurements using 2',7'-dichlorodihydrofluorescein diacetate. Platelet function was analyzed by optical aggregometry using the agonists adenosine diphosphate, collagen, ristocetin and epinephrine. The expression of platelet receptors CD41/CD61, CD42a/CD42b and CD62P/CD63 was evaluated by flow cytometry. Platelet membrane fluidity in patients with MDS was similar to that of healthy volunteers and did not vary according to the risk category. Patients with MDS had increased platelet ROS production compared with the control group without statistical correlation with membrane fluidity. We found a negative correlation of ROS levels with the severity of anemia (R =  -0.587, P = 0.017). Platelet response was reduced in patients with MDS compared with volunteers, for all reagents. The response was different according to the risk category only in case of ristocetin or collagen. Patients with anemia presented a decreased platelet aggregation induced by collagen or ristocetin (collagen: R = 0.395, P = 0.003; ristocetin: R = 0.420, P = 0.002). The membrane fluidity of platelets from MDS patients appeared unmodified, but the ROS production was increased in all risk categories of MDS. The levels of ROS were correlated with the degree of anemia, which, in turn, had a negative impact on the platelet aggregation function induced by collagen or ristocetin.

Stretching of red blood cells using an electro-optics trap.

The stretching stiffness of Red Blood Cells (RBCs) was investigated using a combination of an AC dielectrophoretic apparatus and a single-beam optical tweezer. The experiments were performed at 10 MHz, a frequency high enough to avoid conductivity losses, but below the second turnover point between positive and negative dielectrophoresis. By measuring the geometrical parameters of single healthy human RBCs as a function of the applied voltage, the elastic modulus of RBCs was determined (µ = 1.80 ± 0.5 µN/m) and compared with similar values of the literature got by other techniques. The method is expected to be an easy-to-use, alternative tool to determine the mechano-elastic properties of living cells, and, on this basis, to distinguish healthy and diseased cells.

Iron oxide nanoparticles modulate the interaction of different antibiotics with cellular membranes.

The interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging and drug/gene delivery. These applications require a firm control over nanoparticle-cell interactions, which are mainly dictated by surface properties of the nanoparticles. The aim of this study was to investigate the interaction of Fe3O4 nanoparticles functionalized with several wide use antibiotics with opossum kidney (OK) cellular membranes in order to reveal changes in the membrane organization at different temperatures. We also investigated the in vivo biodistribution of the tested nanoparticles in a mouse model. Our results showed that, at low temperatures (31-35°C), plain Fe3O4 nanoparticles induced a drop of the membrane fluidity, while at physiological or higher temperatures (37-39°C) the membrane fluidity was increased. On the other hand, when nanoparticles functionalized with the tested antibiotics were used, we observed that the effect was opposite as compared to control Fe3O4 nanoparticles. Although most of antibiotics, used as plain solutions or linked on magnetite nanoparticles, proved heterogeneous effect on in vitro OK cells membrane fluidity, the aminoglycosides streptomycin and neomycin, used both as plain solutions and also combined with nanoparticles kept the same effect in all experimental conditions, increasing the membrane fluidity of OK cells plasma membrane. In vivo results showed that the antibiotic functionalized nanoparticles have a similar biodistribution pattern within the mouse body, being transported through the blood flow and entering the macrophages through endocytosis. Functionalized magnetite nanoparticles manifested a preferential biodistribution pattern, clustering within the lungs and spleen of treated mice. These results demonstrate that antibiotics manifest a different effect on plasma membrane fluidity depending on their type and temperature. Magnetite nanoparticles may interfere with antibiotic-cellular interactions by changing the plasma membrane fluidity. The fact that the antibiotic functionalized magnetite nanoparticles have a similar biodistribution pattern, are transported through the blood flow, and they increase the cellular uptake of the drug, suggest that they may be used for further studies aiming to develop personalized targeted delivery and controlled release nanoshuttles for treating localized and systemic infections.

Correlation of the intracellular reactive oxygen species levels with textural properties of functionalized mesostructured silica.

Mesostructured silica is frequently used in biomedical applications, being considered nontoxic and biocompatible material, suitable for the development of drug delivery systems (DDS). Four functionalized MCM-41 silica materials with hydrophobic (methyl and vinyl) and hydrophilic (3-aminopropyl and 3-mercaptopropyl) groups were obtained by post-synthesis functionalization and characterized by small-angle X-ray diffraction, infrared spectroscopy, thermal analysis, and nitrogen adsorption-desorption isotherms. The main structural and textural parameters of the obtained silica were determined. The effect of the functionalized silica on fibroblast (NIH3T3) and melanocyte cells (B16F10) was studied with respect to the proliferation rate and the levels of reactive oxygen species (ROS). It was found that the textural properties of all samples influenced the levels of intracellular ROS and consequently, the proliferation rate. Both, healthy and malignant cells exhibited linear dependence of ROS levels with the specific surface area values, but with different response. The contribution of the methyl functionalized silica to the ROS level is apart to the general trend.

Assessment of changes in membrane properties of platelets from patients with chronic myeloid leukaemia in different stages of the disease.

Patients with chronic myeloproliferative leukemia (CML) have frequent haemorrhage and/or thrombosis in their medical history. The mechanisms of these major and life-threatening complications remain unclear. Membrane organization influences many of the unique cellular functions and is strongly correlated, among other factors, to the membrane lipid composition; it may be evaluated by following up the membrane fluidity and aggregation properties of the platelet. In this study, we evaluated the platelet aggregation, the expression of platelet surface receptors, the membrane fluidity (as evaluated by fluorescence anisotropy) and its correlation to reactive oxygen species (ROS) production, in patients with chronic myeloid leukaemia (CML). It was found that the patients in accelerated and blastic phase of CML present an altered platelet aggregation response to all reagents except for ristocetin as compared with chronic phase group, which shows only epinephrine-altered response. We also found that BCR/ABL transcript leads to higher levels of ROS in accelerated and blastic CML phases. Patients without molecular remission have lower platelet membrane fluidity. We obtained a positive correlation between ROS level and membrane fluorescence anisotropy changes. The CD41 expression was decreased in CML patients and P selectin expression was found to be higher in these patients than in healthy volunteers. Platelets of CML patients have altered aggregation parameters in accelerated and blastic phases, in which BCR/ABL transcript level is increased. The increased level of ROS in CML patients without molecular remission is associated with a decrease in fluidity of platelet membrane and expression of CD41/CD61 receptors. These findings may contribute to understanding the mechanism of the altered platelet response reported in CML patients.

Changes of cell electrical parameters induced by electroporation. A dielectrophoresis study.

Dielectrophoresis was employed to distinguish the electroporated from non-electroporated cells. It was found that the electric field frequency at which cells change the direction of their movement (the crossover frequency f(CO)) is higher when cells are electroporated. The contribution to the cell dielectrophoretic behavior of four electric and geometrical cell parameters was analyzed using a single shell model. f(CO) measurements were performed in media with conductivities of 0.001-0.09S/m, on B16F10 cells which were electroporated in a Mannitol solution (0.001S/m), using rectangular or exponential pulses. The control cells' f(CO) was found in a domain of 2 to 105 kHz, while the electroporated cells' f(CO) was in a domain of 5 to 350 kHz, depending on the external media conductivities. At exterior conductivities above ~0.02S/m, f(CO) of electroporated cells became significantly higher compared to controls. Even though the possible contribution of membrane permittivity to explain the observed f(CO) shift toward higher values cannot be excluded, the computations highlight the fact that the variation of cytosol conductivity might be the major contributor to the dielectrophoretic behavior change. Our experimental observations can be described by considering a linear dependence of electroporated cells' cytosol conductivity against external conductivity.