Red Blood Cell Membrane Fluctuations Research Articles

High spatial and temporal resolution synthetic aperture phase microscopy

A new optical microscopy technique, termed high spatial and temporal resolution synthetic aperture phase microscopy (HISTR-SAPM), is proposed to improve the lateral resolution of wide-field coherent imaging. Under plane wave illumination, the resolution is increased by twofold to around 260 nm, while achieving millisecond-level temporal resolution. In HISTR-SAPM, digital micromirror devices are used to actively change the sample illumination beam angle at high speed with high stability. An off-axis interferometer is used to measure the sample scattered complex fields, which are then processed to reconstruct high-resolution phase images. Using HISTR-SAPM, we are able to map the height profiles of subwavelength photonic structures and resolve the period structures that have 198 nm linewidth and 132 nm gap (i.e., a full pitch of 330 nm). As the reconstruction averages out laser speckle noise while maintaining high temporal resolution, HISTR-SAPM further enables imaging and quantification of nanoscale dynamics of live cells, such as red blood cell membrane fluctuations and subcellular structure dynamics within nucleated cells. We envision that HISTR-SAPM will broadly benefit research in material science and biology.

Portable quantitative phase microscope for material metrology and biological imaging

Quantitative phase microscopy (QPM) has emerged as an important tool for material metrology and biological imaging. For broader adoption in those applications, we have proposed and demonstrated a new portable off-axis QPM method, which works in both transmission and reflection modes to meet different sample measurement requirements. The temporal and spatial sensitivities of our system, as quantified by optical path-length difference values, are 0.65 nm and 1.04 nm, respectively. To demonstrate its applicability for a wide range of applications, we deployed our system for profiling transistor gold electrode samples, observing red blood cell membrane fluctuations, imaging living cells flowing in a microfluidic chip, etc. Our portable QPM system has a low-cost design and involves a simple and robust phase-retrieval algorithm that we envision will allow for broader deployment at different environmental settings, including in resource-limited sites and integration with other metrology or imaging modalities.

Antioxidant effect of resveratrol in single red blood cells measured by thermal fluctuation spectroscopy

The human red blood cell (RBC) membrane has significant elastic capabilities which can be described measuring typical membrane edge fluctuations and mechanical properties by optical techniques. The RBC elastic properties can be affected by changes in the surrounding media. In an attempt to elucidate the molecular mechanisms of the interaction of resveratrol with the red cell membrane and of its antioxidant capacity the changes in mechanical properties of the RBC membrane were analyzed. These studies were carried out through measurements of RBC membrane fluctuations in the presence of the oxidant agent HClO using thermal fluctuation spectroscopy (TFS). The observed results showed that the elastic capabilities of RBC changed with low concentration of hypochlorous acid but without morphological changes. However, in the presence of resveratrol the deformation and decrease of elastic capabilities induced by HClO on RBC decreased. These in vitro results demonstrated the protective effect of RV against the detrimental effects triggered by HClO upon human erythrocytes.

Digital micromirror device-based common-path quantitative phase imaging.

We propose a novel common-path quantitative phase imaging (QPI) method based on a digital micromirror device (DMD). The DMD is placed in a plane conjugate to the objective back-aperture plane for the purpose of generating two plane waves that illuminate the sample. A pinhole is used in the detection arm to filter one of the beams after sample to create a reference beam. Additionally, a transmission-type liquid crystal device, placed at the objective back-aperture plane, eliminates the specular reflection noise arising from all the "off" state DMD micromirrors, which is common in all DMD-based illuminations. We have demonstrated high sensitivity QPI, which has a measured spatial and temporal noise of 4.92 nm and 2.16 nm, respectively. Experiments with calibrated polystyrene beads illustrate the desired phase measurement accuracy. In addition, we have measured the dynamic height maps of red blood cell membrane fluctuations, showing the efficacy of the proposed system for live cell imaging. Most importantly, the DMD grants the system convenience in varying the interference fringe period on the camera to easily satisfy the pixel sampling conditions. This feature also alleviates the pinhole alignment complexity. We envision that the proposed DMD-based common-path QPI system will allow for system miniaturization and automation for a broader adaption.

The Effects of Ethanol on the Morphological and Biochemical Properties of Individual Human Red Blood Cells.

Here, we report the results of a study on the effects of ethanol exposure on human red blood cells (RBCs) using quantitative phase imaging techniques at the level of individual cells. Three-dimensional refractive index tomograms and dynamic membrane fluctuations of RBCs were measured using common-path diffraction optical tomography, from which morphological (volume, surface area, and sphericity); biochemical (hemoglobin (Hb) concentration and Hb content); and biomechanical (membrane fluctuation) parameters were retrieved at various concentrations of ethanol. RBCs exposed to the ethanol concentration of 0.1 and 0.3% v/v exhibited cell sphericities higher than those of normal cells. However, mean surface area and sphericity of RBCs in a lethal alcoholic condition (0.5% v/v) are not statistically different with those of healthy RBCs. Meanwhile, significant decreases of Hb content and concentration in RBC cytoplasm at the lethal condition were observed. Furthermore, dynamic fluctuation of RBC membranes increased significantly upon ethanol treatments, indicating ethanol-induced membrane fluidization.

Red Blood Cell Membrane Fluctuations and their Mechanisms: Passive Versus Active

Red blood cells (RBCs) are soft and flexible biconcave discs, which are able to pass through capillaries with diameters several times smaller than the RBC size. The RBC deformability also results in noticeable membrane fluctuations, which must be correlated with RBC membrane properties. However, it is still under debate whether RBC membrane fluctuations are simply passive thermal undulations or whether a red cell also experiences active fluctuations which are driven by a metabolic activity or other cell processes. We will present direct evidence that the RBC undulations are not solely passive thermal fluctuations, which has been obtained from a set of different experiments and simulations using a high spatiotemporal resolution: from 10 microseconds to several seconds in time and up to 20 nanometers in space. Experimental results show a violation of the fluctuation-dissipation theorem (FDT) for freshly prepared RBCs indicating the existence of active processes. However, the FDT is satisfied for starved cells demonstrating that the membrane fluctuations are passive when the energy supply is absent. Experiments also show a considerable change in the fluctuation amplitudes for fresh and starved cells. Subsequently, we perform simulations which fully mimic and quantify the experiments. We are able to quantitatively extract RBC membrane properties including shear elasticity, bending rigidity, and membrane viscosity. Furthermore, we test several models for active fluctuations, which mimic different possible mechanisms including spectrin network remodeling, ion pumps, and change in the spontaneous membrane curvature. Simulation results agree well with experimental data and suggest that several processes mentioned above may contribute to active RBC fluctuations. We will discuss which processes are more likely to take place.

Optical measurement of biomechanical properties of individual erythrocytes from a sickle cell patient

Sickle cell disease (SCD) is characterized by the abnormal deformation of red blood cells (RBCs) in the deoxygenated condition, as their elongated shape leads to compromised circulation. The pathophysiology of SCD is influenced by both the biomechanical properties of RBCs and their hemodynamic properties in the microvasculature. A major challenge in the study of SCD involves accurate characterization of the biomechanical properties of individual RBCs with minimum sample perturbation. Here we report the biomechanical properties of individual RBCs from a SCD patient using a non-invasive laser interferometric technique. We optically measure the dynamic membrane fluctuations of RBCs. The measurements are analyzed with a previously validated membrane model to retrieve key mechanical properties of the cells: bending modulus; shear modulus; area expansion modulus; and cytoplasmic viscosity. We find that high cytoplasmic viscosity at ambient oxygen concentration is principally responsible for the significantly decreased dynamic membrane fluctuations in RBCs with SCD, and that the mechanical properties of the membrane cortex of irreversibly sickled cells (ISCs) are different from those of the other types of RBCs in SCD.

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.

Coherence properties of red blood cell membrane motions

We use a highly sensitive, noncontact, optical interferometric technique to quantify the red blood cell membrane fluctuations at the nanometer and millisecond scales. The results reveal significant properties of both temporal and spatial coherence associated with the membrane dynamics. We show that these correlations can be accounted for by the viscoelastic properties of the cell membrane. From this measurement, we extract the loss and storage moduli associated with the membrane and find a crossover frequency at which the buffer viscosity seems to become dominant.

Red Blood Cell Membrane Fluctuations and Shape Controlled by ATP-Induced Cytoskeletal Defects

We show theoretically how adenosine 5′-triphosphate (ATP)-induced dynamic dissociations of spectrin filaments (from each other and from the membrane) in the cytoskeleton network of red blood cells (RBC) can explain in a unified manner both the measured fluctuation amplitude as well as the observed shape transformations as a function of intracellular ATP concentration. Static defects can be induced by external stresses such as those present when RBCs pass through small capillaries. We suggest that the partially freed actin at these defect sites may explain the activation of the CFTR membrane-bound protein and the subsequent release of ATP by RBCs subjected to deformations. Our theoretical predictions can be tested by experiments that measure the correlation between variations in the binding of actin to spectrin, the activity of CFTR, and the amount of ATP released.