Interferometric phase-dispersion microscopy

We describe a new scanning microscopy technique, phase-dispersion microscopy (PDM). The technique is based on measuring the phase difference between the fundamental and the second-harmonic light in a novel interferometer. PDM is highly sensitive to subtle refractive-index differences that are due to dispersion (differential optical path sensitivity, 5 nm). We apply PDM to measure minute amounts of DNA in solution and to study biological tissue sections. We demonstrate that PDM performs better than conventional phase-contrast microscopy in imaging dispersive and weakly scattering samples

Extracellular matrix remodeling following myocardial infarction influences the therapeutic potential of mesenchymal stem cells

INTRODUCTION: Although stem cell therapy is a promising treatment for myocardial infarction, the minimal functional improvements observed clinically limit its widespread application. A need exists to maximize the therapeutic potential of these stem cells by first understanding what factors within the infarct microenvironment affect their ability to regenerate the necrotic tissue. In this study, we assessed both differentiation capacity and paracrine signaling as a function of extracellular matrix remodeling after myocardial infarction. METHODS: Mechanical and compositional changes to the decellularized infarcted myocardium were characterized to understand how the extracellular environment, specifically, was altered as a function of time after coronary artery ligation in Sprague–Dawley rats. These alterations were first modeled in a polyacrylamide gel system to understand how the variables of composition and stiffness drive mesenchymal stem cell differentiation towards a cardiac lineage. Finally, the paracrine secretome was characterized as a function of matrix remodeling through gene and protein expression and conditioned media studies. RESULTS: The decellularized infarct tissue revealed significant alterations in both the mechanical and compositional properties of the ECM with remodeling following infarction. This altered microenvironment dynamically regulates the potential for early cardiac differentiation. Whereas Nkx2.5 expression is limited in the presence of chronic remodeled matrix of increased stiffness, GATA4 expression is enhanced. In addition, the remodeled matrix promotes the expression of several proangiogenic, prosurvival, antifibrotic, and immunomodulatory growth factors. In particular, an increase in HGF and SDF1 expression and secretion by mesenchymal stem cells can rescue oxidatively stressed cardiomyocytes in vitro. CONCLUSIONS: This study demonstrated that decellularization of diseased tissue allows for the exclusive analysis of the remodeled matrix and its ability to influence significantly the cellular phenotype. Characterization of cell fate as a function of myocardial remodeling following infarction is critical in developing the ideal strategy for cell implantation to maximize tissue regeneration and to ultimately reduce the prevalence and severity of heart failure

Automated Biochemical, Morphological, and Organizational Assessment of Precancerous Changes from Endogenous Two-Photon Fluorescence Images

Multi-photon fluorescence microscopy techniques allow for non-invasive interrogation of live samples in their native environment. These methods are particularly appealing for identifying pre-cancers because they are sensitive to the early changes that occur on the microscopic scale and can provide additional information not available using conventional screening techniques.In this study, we developed novel automated approaches, which can be employed for the real-time analysis of two-photon fluorescence images, to non-invasively discriminate between normal and pre-cancerous/HPV-immortalized engineered tissues by concurrently assessing metabolic activity, morphology, organization, and keratin localization. Specifically, we found that the metabolic activity was significantly enhanced and more uniform throughout the depths of the HPV-immortalized epithelia, based on our extraction of the NADH and FAD fluorescence contributions. Furthermore, we were able to separate the keratin contribution from metabolic enzymes to improve the redox estimates and to use the keratin localization as a means to discriminate between tissue types. To assess morphology and organization, Fourier-based, power spectral density (PSD) approaches were employed. The nuclear size distribution throughout the epithelial depths was quantified by evaluating the variance of the corresponding spatial frequencies, which was found to be greater in the normal tissue compared to the HPV-immortalized tissues. The PSD was also used to calculate the Hurst parameter to identify the level of organization in the tissues, assuming a fractal model for the fluorescence intensity fluctuations within a field. We found the range of organization was greater in the normal tissue and closely related to the level of differentiation.A wealth of complementary morphological, biochemical and organizational tissue parameters can be extracted from high resolution images that are acquired based entirely on endogenous sources of contrast. They are promising diagnostic parameters for the non-invasive identification of early cancerous changes and could improve significantly diagnosis and treatment for numerous patients

Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields

<p>Abstract</p> <p>Background</p> <p>Electric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing. The application of electric fields offers substantial therapeutic potential, while optimal dosing regimens and the underlying mechanisms responsible for the positive clinical impact are poorly understood.</p> <p>Methods</p> <p>The purpose of this study was to track the differentiation profile and stress response of human bone marrow derived mesenchymal stem cells (hMSCs) undergoing osteogenic differentiation during exposure to a 20 mV/cm, 60 kHz electric field. Morphological and biochemical changes were imaged using endogenous two-photon excited fluorescence (TPEF) and quantitatively assessed through eccentricity calculations and extraction of the redox ratio from NADH, FAD and lipofuscin contributions. Real time reverse transcriptase-polymerase chain reactions (RT-PCR) were used to track osteogenic differentiation markers, namely alkaline phosphatase (ALP) and collagen type 1 (col1), and stress response markers, such as heat shock protein 27 (hsp27) and heat shock protein 70 (hsp70). Comparisons of collagen deposition between the stimulated hMSCs and controls were examined through second harmonic generation (SHG) imaging.</p> <p>Results</p> <p>Quantitative differences in cell morphology, as described through an eccentricity ratio, were found on days 2 and days 5 (p < 0.05) in samples exposed to the electric field. A delayed but two fold increase in ALP and col1 transcript was detected by week 2 (p < 0.05) in differentiating hMSCs exposed to an electric field in comparison to the nonstimulated controls. Upregulation in stress marker, hsp27, and type 1 collagen deposition were correlated with this response. Increases in NADH, FAD, and lipofuscin were traced in the stimulation group during the first week of field exposure with differences statistically significant on day 10 (p < 0.05). Changes in hsp27 expression correlate well with changes in lipofuscin detected in the stimulation group, suggesting a connection with oxidative stress. Both differentiation factors and electrical stimulation improved hMSC differentiation potential to bone based on calcium deposition on day 28.</p> <p>Conclusions</p> <p>Electrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes.</p

Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes. © 2012 Jin et al

From single cells to tissues: interactions between the matrix and human breast cells in real time.

International audienceMammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma--epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis. The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology. Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs

Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner

Nonlinear Optical Microscopy for Histology of Fresh Normal and Cancerous Pancreatic Tissues

BACKGROUND: Pancreatic cancer is a lethal disease with a 5-year survival rate of only 1-5%. The acceleration of intraoperative histological examination would be beneficial for better management of pancreatic cancer, suggesting an improved survival. Nonlinear optical methods based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) of intrinsic optical biomarkers show the ability to visualize the morphology of fresh tissues associated with histology, which is promising for real-time intraoperative evaluation of pancreatic cancer. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate whether the nonlinear optical imaging methods have the ability to characterize pancreatic histology at cellular resolution, we studied different types of pancreatic tissues by using label-free TPEF and SHG. Compared with other routine methods for the preparation of specimens, fresh tissues without processing were found to be most suitable for nonlinear optical imaging of pancreatic tissues. The detailed morphology of the normal rat pancreas was observed and related with the standard histological images. Comparatively speaking, the preliminary images of a small number of chemical-induced pancreatic cancer tissues showed visible neoplastic differences in the morphology of cells and extracellular matrix. The subcutaneous pancreatic tumor xenografts were further observed using the nonlinear optical microscopy, showing that most cells are leucocytes at 5 days after implantation, the tumor cells begin to proliferate at 10 days after implantation, and the extracellular collagen fibers become disordered as the xenografts grow. CONCLUSIONS/SIGNIFICANCE: In this study, nonlinear optical imaging was used to characterize the morphological details of fresh pancreatic tissues for the first time. We demonstrate that it is possible to provide real-time histological evaluation of pancreatic cancer by the nonlinear optical methods, which present an opportunity for the characterization of the progress of spontaneous pancreatic cancer and further application in a non-invasive manner