Alexander Barbul

Comparative study of human erythrocytes by digital holographic microscopy, confocal microscopy, and impedance volume analyzer.

Red blood cell (RBC) parameters such as morphology, volume, refractive index, and hemoglobin content are of great importance for diagnostic purposes. Existing approaches require complicated calibration procedures and robust cell perturbation. As a result, reference values for normal RBC differ depending on the method used. We present a way for measuring parameters of intact individual RBCs by using digital holographic microscopy (DHM), a new interferometric and label‐free technique with nanometric axial sensitivity. The results are compared with values achieved by conventional techniques for RBC of the same donor and previously published figures. A DHM equipped with a laser diode (λ = 663 nm) was used to record holograms in an off‐axis geometry. Measurements of both RBC refractive indices and volumes were achieved via monitoring the quantitative phase map of RBC by means of a sequential perfusion of two isotonic solutions with different refractive indices obtained by the use of Nycodenz (decoupling procedure). Volume of RBCs labeled by membrane dye Dil was analyzed by confocal microscopy. The mean cell volume (MCV), red blood cell distribution width (RDW), and mean cell hemoglobin concentration (MCHC) were also measured with an impedance volume analyzer. DHM yielded RBC refractive index n = 1.418 ± 0.012, volume 83 ± 14 fl, MCH = 29.9 pg, and MCHC 362 ± 40 g/l. Erythrocyte MCV, MCH, and MCHC achieved by an impedance volume analyzer were 82 fl, 28.6 pg, and 349 g/l, respectively. Confocal microscopy yielded 91 ± 17 fl for RBC volume. In conclusion, DHM in combination with a decoupling procedure allows measuring noninvasively volume, refractive index, and hemoglobin content of single‐living RBCs with a high accuracy.

Spatial analysis of erythrocyte membrane fluctuations by digital holographic microscopy

Red blood cell (RBC) membrane fluctuations provide important insights into cell states. We present a spatial analysis of red blood cell membrane fluctuations by using digital holographic microscopy (DHM). This interferometric and dye-free technique, possessing nanometric axial and microsecond temporal sensitivities enables to measure cell membrane fluctuations (CMF) on the whole cell surface. DHM acquisition is combined with a model which allows extracting the membrane fluctuation amplitude, while taking into account cell membrane topology. Uneven distribution of CMF amplitudes over the RBC surface is observed, showing maximal values in a ring corresponding to the highest points on the RBC torus as well as in some scattered areas in the inner region of the RBC. CMF amplitudes of 35.9+/-8.9 nm and 4.7+/-0.5 nm (averaged over the cell surface) were determined for normal and ethanol-fixed RBCs, respectively.

Terahertz Radiation Increases Genomic Instability in Human Lymphocytes

Abstract Korenstein-Ilan, A., Barbul, A., Hasin, P., Eliran, A., Gover, A. and Korenstein, R. Terahertz Radiation Increases Genomic Instability in Human Lymphocytes. Radiat. Res. 170, 224–234 (2008). Terahertz radiation is increasingly being applied in new and evolving technologies applied in areas such as homeland security and medical imaging. Thus a timely assessment of the potential hazards and health effects of occupational and general population exposure to THz radiation is required. We applied continuous-wave (CW) 0.1 THz radiation (0.031 mW/ cm2) to dividing lymphocytes for 1, 2 and 24 h and examined the changes in chromosome number of chromosomes 1, 10, 11 and 17 and changes in the replication timing of their centromeres using interphase fluorescence in situ hybridization (FISH). Chromosomes 11 and 17 were most vulnerable (about 30% increase in aneuploidy after 2 and 24 h of exposure), while chromosomes 1 and 10 were not affected. We observed changes in the asynchronous mode of replication of centromeres 11, 17 and 1 (by 40%) after 2 h of exposure and of all four centromeres after 24 h of exposure (by 50%). It is speculated that these effects are caused by radiation-induced low-frequency collective vibrational modes of proteins and DNA. Our results demonstrate that exposure of lymphocytes in vitro to a low power density of 0.1 THz radiation induces genomic instability. These findings, if verified, may suggest that such exposure may result in an increased risk of cancer.

Increased Levels of Numerical Chromosome Aberrations after In Vitro Exposure of Human Peripheral Blood Lymphocytes to Radiofrequency Electromagnetic Fields for 72 Hours

Abstract Mazor, R., Korenstein-Ilan, A., Barbul, A., Eshet, Y., Shahadi, A., Jerby, E. and Korenstein, R. Increased Levels of Numerical Chromosome Aberrations after In Vitro Exposure of Human Peripheral Blood Lymphocytes to Radiofrequency Electromagnetic Fields for 72 Hours. Radiat. Res. 169, 28–37 (2008). We investigated the effects of 72 h in vitro exposure of 10 human lymphocyte samples to radiofrequency electromagnetic fields (800 MHz, continuous wave) on genomic instability. The lymphyocytes were exposed in a specially designed waveguide resonator at specific absorption rates (SARs) of 2.9 and 4.1 W/kg in a temperature range of 36–37°C. The induced aneuploidy of chromosomes 1, 10, 11 and 17 was determined by interphase FISH using semi-automated image analysis. We observed increased levels of aneuploidy depending on the chromosome studied as well as on the level of exposure. In chromosomes 1 and 10, there was increased aneuploidy at the higher SAR, while for chromosomes 11 and 17, the increases were observed only for the lower SAR. Multisomy (chromosomal gains) appeared to be the primary contributor to the increased aneuploidy. The effect of temperature on the level of aneuploidy was examined over the range of 33.5–40°C for 72 h with no statistically significant difference in the level of aneuploidy compared to 37°C. These findings suggest the possible existence of an athermal effect of RF radiation that causes increased levels of aneuploidy. These results contribute to the assessment of potential health risks after continuous chronic exposure to RF radiation at SARs close to the current levels set by ICNIRP guidelines.

Ca2+ promotes erythrocyte band 3 tyrosine phosphorylation via dissociation of phosphotyrosine phosphatase from band 3.

The anion-exchange band 3 protein is the main erythrocyte protein that is phosphorylated by protein tyrosine kinase (PTK). We have previously identified a band 3-associated phosphotyrosine phosphatase (PTP) that is normally highly active and prevents the accumulation of band 3 phosphotyrosine. Band 3 tyrosine phosphorylation can be induced by inhibition of PTP (vanadate, thiol oxidation), activation of PTK (hypertonic NaCl) or intracellular increased Ca(2+) (mechanism unknown). We now show that there is inhibition of dephosphorylation of band 3 in Ca(2+)/ionophore-treated erythrocytes and in membranes isolated from the treated cells. These membranes exhibit phosphatase activity upon the addition of exogenous substrate. Dephosphorylation of the endogenous substrate (band 3) can be activated in these membranes by the addition of Mg(2+). Thus the inability of PTP to dephosphorylate the band 3 phosphotyrosine is not due to inhibition of the enzyme itself. Ca(2+) rise in the erythrocyte causes dissociation of PTP from band 3, thus leaving the kinase unopposed. This is shown by a significant diminution in band 3/PTP co-precipitation. Addition of Mg(2+) to these membranes leads to reassociation of band 3 with PTP. The Ca(2+)-induced inhibition of band 3 dephosphorylation may be due to Ca(2+)-dependent alterations in membrane components and structure, affecting the interaction of band 3 with PTP. The Ca(2+)-induced tyrosine phosphorylation, involving an apparent PTP inhibition via dissociation from the substrate, may play a role in signal transduction pathways and in certain pathological disorders associated with increased cell Ca(2+).

Optical phase nanoscopy in red blood cells using low-coherence spectroscopy

Abstract. We propose a low-coherence spectral-domain phase microscopy (SDPM) system for accurate quantitative phase measurements in red blood cells (RBCs) for the prognosis and monitoring of disease conditions that affect the visco-elastic properties of RBCs. Using the system, we performed time-recordings of cell membrane fluctuations, and compared the nano-scale fluctuation dynamics of healthy and glutaraldehyde-treated RBCs. Glutaraldehyde-treated RBCs possess lower amplitudes of fluctuations, reflecting an increased membrane stiffness. To demonstrate the ability of our system to measure fluctuations of lower amplitudes than those measured by the commonly used holographic phase microscopy techniques, we also constructed wide-field digital interferometry (WFDI) system and compared the performances of both systems. Due to its common-path geometry, the optical-path-delay stability of SDPM was found to be less than 0.3 nm in liquid environment, at least three times better than WFDI under the same conditions. In addition, due to the compactness of SDPM and its inexpensive and robust design, the system possesses a high potential for clinical applications.

Electrofusion of fibroblasts on the porous membrane

Electric fusion of cells is usually performed in two steps: the first is the creation of tight intercellular contact, the second is an application of electric pulses which induce membrane fusion proper. In the present work a new technique of cell electrofusion on the porous film is described. It consists of preliminary cultivation of cell monolayer on the porous film (protein-coated cellophane). Then cells of the same or any other type are added from above to form a second cell layer upon the first one. The pulses of the electric field are applied normally to the plane of the double cell layer to induce cell fusion. After pulse application a picture of mass polynucleation was observed. At the same time we did not obtain fusion of L cells by means of dielectrophoretic electrofusion technique. This difference in efficiency could be explained by the formation of broad zones of membrane contact between the cells adherent to the film, while during intensive dielectrophoresis only the point contacts were revealed. The high-conducting medium for electric treatment providing an efficient fusion on the film and high cell viability was composed. Neither cytochalasin B nor colcemid affected cell fusion noticeably; however the sodium azide (added with 2-deoxyglucose) inhibited fusion completely. The short hypotonic shock after electric treatment enhanced the rate of polycaryon formation.

Low electric fields induce ligand-independent activation of EGF receptor and ERK via electrochemical elevation of H(+) and ROS concentrations.

Physiological electric fields are involved in many biological processes and known to elicit their effects during long exposures ranging from a few hours to days. Following exposure to electric fields of physiological amplitude, epidermal growth factor receptor (EGFR) was demonstrated to be redistributed and upregulated with further intracellular signaling such as the MAPK signaling cascade. In our study we demonstrated EGFR activation and signaling induced by short train of pulsed low electric field (LEF) (10V/cm, pulse-width 180μs, 500Hz, 2min) in serum-free medium, following 24-hour starvation, and in the absence of exogenous EGF ligand, suggesting a ligand-independent pathway for EGFR activation. This ligandless activation was further confirmed by using neutralizing antibodies (LA1) that block the EGFR ligand-binding site. EGFR activation was found to be EGFR kinase dependent, yet with no dimerization following exposure to LEF. ERK activation was found to be mainly a result of EGFR downstream signaling though it partially occurred via EGFR-independent way. We demonstrate that reactive oxygen species and especially decrease in pH generated during exposure to LEF are involved in EGFR ligandless activation. We propose a possible mechanism for the LEF-induced EGFR ligand-independent activation and show activation of other receptor tyrosine kinases following exposure to LEF.

Erythrocytes volume and refractive index measurement with a digital holographic microscope

Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram, quantitative phase image of living cell with interferometric accuracy. Specifically the optical phase shift induced by the specimen on the transmitted wave front can be regarded as a powerful endogenous contrast agent, depending on both the thickness and the refractive index of the sample. We have recently proposed a new and efficient decoupling procedure allowing to directly obtain separate measurements of the thickness and the integral refractive index of a given living cell. Consequently, it has been possible to accurately measure (with a precision of 0.0003) the mean refractive index and the volume of living erythrocytes. Here, application of this decoupling procedure on erythrocyte allows to measure a refractive index of 1.40 and a mean volume of about 106 μm3.

Optical phase measurements in red blood cells using low-coherence spectroscopy

We demonstrate the use of a low-coherence spectral-domain phase microscopy (SDPM) system for accurate quantitative phase measurements in red blood cells (RBCs) for the prognosis and monitoring of disease conditions that affect the visco-elastic properties of RBCs. Using the system, we performed time-recordings of cell membrane fluctuations, and compared the nano-scale fluctuation dynamics of healthy and glutaraldehyde-treated RBCs. Glutaraldehyde-treated RBCs possess a lower amplitude of fluctuations reflecting an increased membrane stiffness. To demonstrate the ability of our system to measure fluctuations of lower amplitudes than those measured by the commonly used holographic phase microscopy techniques, we also constructed a wide-field digital interferometric microscope and compared the performances of the two systems. Due to its common-path geometry, the optical-path-delay stability of SDPM was found to be less than 0.3nm in liquid environment, at least three times better than in holographic phase microscopy under the same conditions. In addition, due to the compactness of SDPM and its inexpensive and robust design, the system possesses a high potential for clinical applications.