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An increase of oxygen saturation within blood bags and metabolic dysregulation occur during storage of red blood cells (RBCs). It leads to the gradual exhaustion of RBC antioxidant protective system and, consequently, to a deleterious state of oxidative stress that plays a major role in the apparition of the so-called storage lesions. The present study describes the use of a test (called TSOX) based on fluorescence and label-free morphology readouts to simply and quickly evaluate the oxidant and antioxidant properties of various compounds in controlled conditions. Here, TSOX was applied to RBCs treated with four antioxidants (ascorbic acid, uric acid, trolox and resveratrol) and three oxidants (AAPH, diamide and H2O2) at different concentrations. Two complementary readouts were chosen: first, where ROS generation was quantified using DCFH-DA fluorescent probe, and second, based on digital holographic microscopy that measures morphology alterations. All oxidants produced an increase of fluorescence, whereas H2O2 did not visibly impact the RBC morphology. Significant protection was observed in three out of four of the added molecules. Of note, resveratrol induced diamond-shape "Tirocytes". The assay design was selected to be flexible, as well as compatible with high-throughput screening. In future experiments, the TSOX will serve to screen chemical libraries and probe molecules that could be added to the additive solution for RBCs storage.
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Eritrocitos/metabolismo , Microscopía Fluorescente , Imagen Molecular , Oxidantes/metabolismo , Estrés Oxidativo , Antioxidantes/farmacología , Descubrimiento de Drogas , Eritrocitos/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Resveratrol/farmacología , Flujo de TrabajoRESUMEN
In vitro differentiating adipocytes are sensitive to liquid manipulations and have the tendency to float. Assessing adipocyte differentiation using current microscopy techniques involves cell staining and washing, while using flow cytometry involves cell retrieval in suspension. These methods induce biases, are difficult to reproduce, and involve tedious optimizations. In this study, we present digital holographic microscopy (DHM) as a label-free, nonperturbing means to quantify lipid droplets in differentiating adipocytes in a robust medium- to high-throughput manner. Taking advantage of the high refractive index of lipid droplets, DHM can assess the production of intracellular lipid droplets by differences in phase shift in a quantitative manner. Adipocytic differentiation, combined with other morphological features including cell confluence and cell death, was tracked over 6 days in live OP9 mesenchymal stromal cells. We compared DHM with other currently available methods of lipid droplet quantification and demonstrated its robustness with modulators of adipocytic differentiation in a dose-responsive manner. This study suggests DHM as a novel marker-free nonperturbing method to study lipid droplet accumulation and may be envisioned for drug screens and mechanistic studies on adipocytic differentiation.
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Holografía , Gotas Lipídicas/metabolismo , Microscopía , Adipocitos/citología , Adipocitos/metabolismo , Diferenciación Celular , Línea Celular , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Netrins are secreted proteins that direct cell migration and adhesion during development. Netrin-1 binds its receptors deleted in colorectal cancer (DCC) and the UNC5 homologs (UNC5A-D) to activate downstream signaling that ultimately directs cytoskeletal reorganization. To investigate how netrin-1 regulates the dynamic distribution of DCC and UNC5 homologs, we applied fluorescence confocal and total internal reflection fluorescence microscopy, and sliding window temporal image cross correlation spectroscopy, to measure time profiles of the plasma membrane distribution, aggregation state, and interaction fractions of fluorescently tagged netrin receptors expressed in HEK293T cells. Our measurements reveal changes in receptor aggregation that are consistent with netrin-1-induced recruitment of DCC-enhanced green fluorescent protein (EGFP) from intracellular vesicles to the plasma membrane. Netrin-1 also induced colocalization of coexpressed full-length DCC-EGFP with DCC-T-mCherry, a putative DCC dominant negative that replaces the DCC intracellular domain with mCherry, consistent with netrin-1-induced receptor oligomerization, but with no change in aggregation state with time, providing evidence that signaling via the DCC intracellular domain triggers DCC recruitment to the plasma membrane. UNC5B expressed alone was also recruited by netrin-1 to the plasma membrane. Coexpressed DCC and UNC5 homologs are proposed to form a heteromeric netrin-receptor complex to mediate a chemorepellent response. Application of temporal image cross correlation spectroscopy to image series of cells coexpressing UNC5B-mCherry and DCC-EGFP revealed a netrin-1-induced increase in colocalization, with both receptors recruited to the plasma membrane from preexisting clusters, consistent with vesicular recruitment and receptor heterooligomerization. Plasma membrane recruitment of DCC or UNC5B was blocked by application of the netrin-1 VI-V peptide, which fails to activate chemoattraction, or by pharmacological block of Src family kinase signaling, consistent with receptor recruitment requiring netrin-1-activated signaling. Our findings reveal a mechanism activated by netrin-1 that recruits DCC and UNC5B to the plasma membrane.
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Factores de Crecimiento Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Membrana Celular/metabolismo , Receptor DCC , Células HEK293 , Humanos , Receptores de Netrina , Netrina-1 , Transporte de ProteínasRESUMEN
Red blood cell (RBC) phase images that are numerically reconstructed by digital holographic microscopy (DHM) can describe the cell structure and dynamics information beneficial for a quantitative analysis of RBCs. However, RBCs investigated with time-lapse DHM undergo temporal displacements when their membranes are loosely attached to the substrate during sedimentation on a glass surface or due to the microscope drift. Therefore, we need to develop a tracking algorithm to localize the same RBC among RBC image sequences and dynamically monitor its biophysical cell parameters; this information is helpful for studies on RBC-related diseases and drug tests. Here, we propose a method, which is a combination of the mean-shift algorithm and Kalman filter, to track a single RBC and demonstrate that the optical path length of the single RBC can be continually extracted from the tracked RBC. The Kalman filter is utilized to predict the target RBC position in the next frame. Then, the mean-shift algorithm starts execution from the predicted location, and a robust kernel, which is adaptive to changes in the RBC scale, shape, and direction, is designed to improve the accuracy of the tracking. Finally, the tracked RBC is segmented and parameters such as the RBC location are extracted to update the Kalman filter and the kernel function for mean-shift tracking; the characteristics of the target RBC are dynamically observed. Experimental results show the feasibility of the proposed algorithm.
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Rastreo Celular/métodos , Eritrocitos/citología , Holografía/métodos , Microscopía/métodos , Imagen de Lapso de Tiempo/métodos , Algoritmos , Automatización , Humanos , Movimiento (Física) , Factores de TiempoRESUMEN
Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique-spatial intensity distribution analysis (SpIDA)-that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.
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Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Proteína Proteolipídica de la Mielina/química , Multimerización de Proteína , Animales , Artefactos , Células COS , Chlorocebus aethiops , Simulación por Computador , Dimerización , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Microscopía Confocal/métodos , Modelos Biológicos , Mutación , Proteína Proteolipídica de la Mielina/genética , Proteína Proteolipídica de la Mielina/metabolismo , Fotoblanqueo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Análisis de la Célula IndividualRESUMEN
Compounds tested during drug development may have adverse effects on the heart; therefore all new chemical entities have to undergo extensive preclinical assessment for cardiac liability. Conventional intensity-based imaging techniques are not robust enough to provide detailed information for cell structure and the captured images result in low-contrast, especially to cell with semi-transparent or transparent feature, which would affect the cell analysis. In this paper we show, for the first time, that digital holographic microscopy (DHM) integrated with information processing algorithms automatically provide dynamic quantitative phase profiles of beating cardiomyocytes. We experimentally demonstrate that relevant parameters of cardiomyocytes can be obtained by our automated algorithm based on DHM phase signal analysis and used to characterize the physiological state of resting cardiomyocytes. Our study opens the possibility of automated quantitative analysis of cardiomyocyte dynamics suitable for further drug safety testing and compounds selection as a new paradigm in drug toxicity screens.
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ABSTRACT: The process of protein phosphorylation is involved in numerous cell functions. In particular, phosphotyrosine (pY) has been reported to play a role in red blood cell (RBC) functions, including the cytoskeleton organization. During their storage before transfusion, RBCs suffer from storage lesions that affect their energy metabolism and morphology. This study investigated the relationship between pY and the storage lesions. To do so, RBCs were treated (in the absence of calcium) with a protein tyrosine phosphatase inhibitor (orthovanadate [OV]) to stimulate phosphorylation and with 3 selective kinase inhibitors (KIs). Erythrocyte membrane proteins were studied by western blot analyses and phosphoproteomics (data are available via ProteomeXchange with identifier PXD039914) and cell morphology by digital holographic microscopy. The increase of pY triggered by OV treatment (inducing a global downregulation of pS and pT) disappeared during the storage. Phosphoproteomic analysis identified 609 phosphoproteins containing 1752 phosphosites, of which 41 pY were upregulated and 2 downregulated by OV. After these phosphorylation processes, the shape of RBCs shifted from discocytes to spherocytes, and the addition of KIs partially inhibited this transition. The KIs modulated either pY or pS and pT via diverse mechanisms related to cell shape, thereby affecting RBC morphology. The capacity of RBCs to maintain their function is central in transfusion medicine, and the presented results contribute to a better understanding of RBC biology.
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Conservación de la Sangre , Eritrocitos , Humanos , Conservación de la Sangre/métodos , Eritrocitos/metabolismo , Membrana Eritrocítica/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismoRESUMEN
This manuscript proposes a new dual-mode cell imaging system for studying the relationships between calcium dynamics and the contractility process of cardiomyocytes derived from human-induced pluripotent stem cells. Practically, this dual-mode cell imaging system provides simultaneously both live cell calcium imaging and quantitative phase imaging based on digital holographic microscopy. Specifically, thanks to the development of a robust automated image analysis, simultaneous measurements of both intracellular calcium, a key player of excitation-contraction coupling, and the quantitative phase image-derived dry mass redistribution, reflecting the effective contractility, namely, the contraction and relaxation processes, were achieved. Practically, the relationships between calcium dynamics and the contraction-relaxation kinetics were investigated in particular through the application of two drugsânamely, isoprenaline and E-4031âknown to act precisely on calcium dynamics. Specifically, this new dual-mode cell imaging system enabled us to establish that calcium regulation can be divided into two phases, an early phase influencing the occurrence of the relaxation process followed by a late phase, which although not having a significant influence on the relaxation process affects significantly the beat frequency. In combination with cutting-edge technologies allowing the generation of human stem cell-derived cardiomyocytes, this dual-mode cell monitoring approach therefore represents a very promising technique, particularly in the fields of drug discovery and personalized medicine, to identify compounds likely to act more selectively on specific steps that compose the cardiomyocyte contractility.
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Calcio , Células Madre Pluripotentes Inducidas , Humanos , Miocitos Cardíacos , Cinética , Isoproterenol/farmacologíaRESUMEN
Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010). Here, we refine these observations and show that glutamate produces the following three distinct optical responses in mouse primary cortical neurons in culture, predominantly mediated by NMDA receptors: biphasic, reversible decrease (RD) and irreversible decrease (ID) responses. The shape and amplitude of the optical signal were not associated with a particular cellular phenotype but reflected the physiopathological status of neurons linked to the degree of NMDA activity. Thus, the biphasic, RD, and ID responses indicated, respectively, a low-level, a high-level, and an "excitotoxic" level of NMDA activation. Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or potassium-coupled chloride movement strongly modified the phase shift, suggesting an involvement of two neuronal cotransporters, NKCC1 (Na-K-Cl) and KCC2 (K-Cl) in the genesis of the optical signal. This observation is of particular interest since it shows that DHM is the first imaging technique able to monitor dynamically and in situ the activity of these cotransporters during physiological and/or pathological neuronal conditions.
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Holografía , Neuronas/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Simportadores/metabolismo , Agua/metabolismo , Animales , Células Cultivadas , Difusión/efectos de los fármacos , Femenino , Furosemida/farmacología , Holografía/métodos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Microscopía Confocal/métodos , Neuronas/citología , Neuronas/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Quinoxalinas/farmacología , Receptores Ionotrópicos de Glutamato/antagonistas & inhibidores , Procesamiento de Señales Asistido por Computador , Miembro 2 de la Familia de Transportadores de Soluto 12 , Cotransportadores de K ClRESUMEN
Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). The most common mutation, ΔF508, causes retention of CFTR in the endoplasmic reticulum (ER). Some CF abnormalities can be explained by altered Ca(2+) homeostasis, although it remains unknown how CFTR influences calcium signaling. This study examined the novel hypothesis that store-operated calcium entry (SOCE) through Orai1 is abnormal in CF. The significance of Orai1-mediated SOCE for increased interleukin-8 (IL-8) expression in CF was also investigated. CF and non-CF human airway epithelial cell line and primary cells (obtained at lung transplantation) were used in Ca(2+) imaging, electrophysiology, and fluorescence imaging experiments to explore differences in Orai1 function in CF vs. non-CF cells. Protein expression and localization was assessed by Western blots, cell surface biotinylation, ELISA, and image correlation spectroscopy (ICS). We show here that store-operated Ca(2+) entry (SOCE) is elevated in CF human airway epithelial cells (hAECs; ≈ 1.8- and ≈ 2.5-fold for total Ca(2+)(i) increase and Ca(2+) influx rate, respectively, and ≈ 2-fold increase in the I(CRAC) current) and is caused by increased exocytotic insertion (≈ 2-fold) of Orai1 channels into the plasma membrane, which is normalized by rescue of ΔF508-CFTR trafficking to the cell surface. Augmented SOCE in CF cells is a major factor leading to increased IL-8 secretion (≈ 2-fold). CFTR normally down-regulates the Orai1/stromal interaction molecule 1 (STIM1) complex, and loss of this inhibition due to the absence of CFTR at the plasma membrane helps to explain the potentiated inflammatory response in CF cells.
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Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Fibrosis Quística/metabolismo , Interleucina-8/biosíntesis , Secuencia de Bases , Canales de Calcio/genética , Membrana Celular/metabolismo , Células Cultivadas , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Cartilla de ADN/genética , Técnicas de Silenciamiento del Gen , Humanos , Potenciales de la Membrana , Proteínas de la Membrana/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , ARN Interferente Pequeño/genética , Mucosa Respiratoria/metabolismo , Transducción de Señal , Molécula de Interacción Estromal 1RESUMEN
The ionotropic GABAA receptors represent the main target for different groups of widely used drugs having hypnotic and anxiolytic effects. So far, most approaches used to assess GABA activity involve invasive low -throughput electrophysiological techniques or rely on fluorescent dyes, preventing the ability to conduct noninvasive and thus nonperturbing screens. To address this limitation, we have developed an automated marker-free cell imaging method, based on digital holographic microscopy (DHM). This technology allows the automatically screening of compounds in multiple plates without having to label the cells or use special plates. This methodological approach was first validated by screening the GABAA receptor expressed in HEK cells using a selection of active compounds in agonist, antagonist, and modulator modes. Then, in a second blind screen of a library of 3041 compounds (mostly composed of natural products), 5 compounds having a specific agonist action on the GABAA receptor were identified. The hits validated from this unbiased screen were the natural products muscimol, neurosteroid alphaxalone, and three compounds belonging to the avermectin family, all known for having an agonistic effect on the GABAA receptor. The results obtained were exempt from false negatives (structurally similar unassigned hits), and false-positive hits were detected and discarded without the need for performing electrophysiological measurements. The outcome of the screen demonstrates the applicability of our screening by imaging method for the discovery of new chemical structures, particularly regarding chemicals interacting with the ionotropic GABAA receptor and more generally with any ligand-gated ion channels and transporters.
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Agonistas de Receptores de GABA-A/aislamiento & purificación , Antagonistas de Receptores de GABA-A/aislamiento & purificación , Imagen Molecular/métodos , Receptores de GABA-A/genética , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Fenómenos Electrofisiológicos , Agonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/química , Ensayos Analíticos de Alto Rendimiento/métodos , Holografía , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía , Ácido gamma-Aminobutírico/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
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.
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Membrana Eritrocítica/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodos , Adulto , Algoritmos , Diseño de Equipo , Holografía , Humanos , Procesamiento de Imagen Asistido por Computador/instrumentación , Microscopía/instrumentación , Microscopía de Interferencia , RefractometríaRESUMEN
Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.
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Holografía/métodos , Microscopía/métodos , Schizosaccharomyces/citología , Proteínas de Ciclo Celular/genética , Citocinesis/fisiología , Modelos Lineales , Mutación , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
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 (lambda = 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.
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Eritrocitos/citología , Holografía , Microscopía Confocal/métodos , Procesamiento de Señales Asistido por Computador , Impedancia Eléctrica , Índices de Eritrocitos , Volumen de Eritrocitos , Humanos , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
We propose methods to quantitatively calculate the fluctuation rate of red blood cells with nanometric axial and millisecond temporal sensitivity at the single-cell level by using time-lapse holographic cell imaging. For this quantitative analysis, cell membrane fluctuations (CMFs) were measured for RBCs stored at different storage times. Measurements were taken over the whole membrane for both the ring and dimple sections separately. The measurements show that healthy RBCs that maintain their discocyte shape become stiffer with storage time. The correlation analysis demonstrates a significant negative correlation between CMFs and the sphericity coefficient, which characterizes the morphological type of erythrocyte. In addition, we show the correlation results between CMFs and other morphological properties such as projected surface area, surface area, mean corpuscular volume, and mean corpuscular hemoglobin.
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Digital Holographic Microscopy (DHM) is a single shot interferometric technique, which provides quantitative phase images with subwavelength axial accuracy. A short hologram acquisition time (down to microseconds), allows DHM to offer a reduced sensitivity to vibrations, and real time observation is achievable thanks to present performances of personal computers and charge coupled devices (CCDs). Fast dynamic imaging at low-light level involves few photons, requiring proper camera settings (integration time and gain of the CCD; power of the light source) to minimize the influence of shot noise on the hologram when the highest phase accuracy is aimed. With simulated and experimental data, a systematic analysis of the fundamental shot noise influence on phase accuracy in DHM is presented.
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BACKGROUND: Red blood cells collected in citrate-phosphate-dextrose can be stored for up to 42 days at 4 °C in saline-adenine-glucose-mannitol additive solution. During this controlled, but nevertheless artificial, ex vivo ageing, red blood cells accumulate lesions that can be reversible or irreversible upon transfusion. The aim of the present study is to follow several parameters reflecting cell metabolism, antioxidant defences, morphology and membrane dynamics during storage. MATERIALS AND METHODS: Five erythrocyte concentrates were followed weekly during 71 days. Extracellular glucose and lactate concentrations, total antioxidant power, as well as reduced and oxidised intracellular glutathione levels were quantified. Microvesiculation, percentage of haemolysis and haematologic parameters were also evaluated. Finally, morphological changes and membrane fluctuations were recorded using label-free digital holographic microscopy. RESULTS: The antioxidant power as well as the intracellular glutathione concentration first increased, reaching maximal values after one and two weeks, respectively. Irreversible morphological lesions appeared during week 5, where discocytes began to transform into transient echinocytes and finally spherocytes. At the same time, the microvesiculation and haemolysis started to rise exponentially. After six weeks (expiration date), intracellular glutathione was reduced by 25%, reflecting increasing oxidative stress. The membrane fluctuations showed decreased amplitudes during shape transition from discocytes to spherocytes. DISCUSSION: Various types of lesions accumulated at different chemical and cellular levels during storage, which could impact their in vivo recovery after transfusion. A marked effect was observed after four weeks of storage, which corroborates recent clinical data. The prolonged follow-up period allowed the capture of deep storage lesions. Interestingly, and as previously described, the severity of the changes differed among donors.
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Conservación de la Sangre , Envejecimiento Eritrocítico , Eritrocitos/citología , Conservación de la Sangre/métodos , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/patología , Citratos/metabolismo , Eritrocitos/metabolismo , Eritrocitos/patología , Glucosa/metabolismo , Glutatión/metabolismo , Hemólisis , Humanos , Ácido Láctico/metabolismo , Oxidación-Reducción , Estrés OxidativoRESUMEN
PURPOSE: Adaptation to eccentric viewing in subjects with a central scotoma remains poorly understood. The purpose of this study was to analyze the adaptation stages of oculomotor control to forced eccentric reading in normal subjects. METHODS: Three normal adults (25.7 +/- 3.8 years of age) were trained to read full-page texts using a restricted 10 degrees x 7 degrees viewing window stabilized at 15 degrees eccentricity (lower visual field). Gaze position was recorded throughout the training period (1 hour per day for approximately 6 weeks). RESULTS: In the first sessions, eye movements appeared inappropriate for reading, mainly consisting of reflexive vertical (foveating) saccades. In early adaptation phases, both vertical saccade count and amplitude dramatically decreased. Horizontal saccade frequency increased in the first experimental sessions, then slowly decreased after 7 to 15 sessions. Amplitude of horizontal saccades increased with training. Gradually, accurate line jumps appeared, the proportion of progressive saccades increased, and the proportion of regressive saccades decreased. At the end of the learning process, eye movements mainly consisted of horizontal progressions, line jumps, and a few horizontal regressions. CONCLUSIONS: Two main adaptation phases were distinguished: a "faster" vertical process aimed at suppressing reflexive foveation and a "slower" restructuring of the horizontal eye movement pattern. The vertical phase consisted of a rapid reduction in the number of vertical saccades and a rapid but more progressive adjustment of remaining vertical saccades. The horizontal phase involved the amplitude adjustment of horizontal saccades (mainly progressions) to the text presented and the reduction of regressions required.
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Adaptación Ocular/fisiología , Músculos Oculomotores/fisiología , Lectura , Movimientos Sacádicos/fisiología , Adulto , HumanosRESUMEN
This paper presents an optical diffraction tomography technique based on digital holographic microscopy. Quantitative 2-dimensional phase images are acquired for regularly-spaced angular positions of the specimen covering a total angle of pi, allowing to built 3-dimensional quantitative refractive index distributions by an inverse Radon transform. A 20x magnification allows a resolution better than 3 microm in all three dimensions, with accuracy better than 0.01 for the refractive index measurements. This technique is for the first time to our knowledge applied to living specimen (testate amoeba, Protista). Morphometric measurements are extracted from the tomographic reconstructions, showing that the commonly used method for testate amoeba biovolume evaluation leads to systematic under evaluations by about 50%.