Individual Human Red Blood Cells Research Articles

Measuring cell surface area and deformability of individual human red blood cells over blood storage using quantitative phase imaging.

The functionality and viability of stored human red blood cells (RBCs) is an important clinical issue in transfusions. To systematically investigate changes in stored whole blood, the hematological properties of individual RBCs were quantified in blood samples stored for various periods with and without a preservation solution called citrate phosphate dextrose adenine-1 (CPDA-1). With 3-D quantitative phase imaging techniques, the optical measurements for 3-D refractive index (RI) distributions and membrane fluctuations were done at the individual cell level. From the optical measurements, the morphological (volume, surface area and sphericity), biochemical (hemoglobin content and concentration), and mechanical parameters (dynamic membrane fluctuation) were simultaneously quantified to investigate the functionalities and progressive alterations of stored RBCs. Our results show that stored RBCs without CPDA-1 had a dramatic morphological transformation from discocytes to spherocytes within two weeks which was accompanied by significant decreases in cell deformability and cell surface area, and increases in sphericity. However, the stored RBCs with CPDA-1 maintained their morphology and deformability for up to 6 weeks.

Profiling of individual human red blood cells under osmotic stress using defocusing microscopy.

We use a quantitative phase imaging technique, defocusing microscopy (DM), to measure morphological, chemical, and mechanical parameters of individual red blood cells (RBCs) immersed in solutions with different osmolalities. We monitor the RBCs’ radius, volume, surface area, sphericity index, and hemoglobin content and concentration. The complete shape of cells is recovered and the effects of their adhesion to the glass substrate are observed. Finally, membrane fluctuation measurements give us information about the cells deformability.

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.

Three-dimensional refractive index tomograms and deformability of individual human red blood cells from cord blood of newborn infants and maternal blood.

Red blood cells (RBCs) from the cord blood of newborn infants have distinctive functions in fetal and infant development. To systematically investigate the biophysical characteristics of individual cord RBCs in newborn infants, a comparative study was performed on RBCs from the cord blood of newborn infants and from adult mothers or nonpregnant women using optical holographic microtomography. Optical measurements of the distributions of the three-dimensional refractive indices and the dynamic membrane fluctuations of individual RBCs were used to investigate the morphological, biochemical, and mechanical properties of cord, maternal, and adult RBCs at the individual cell level. The volume and surface area of the cord RBCs were significantly larger than those of the RBCs from nonpregnant women, and the cord RBCs had more flattened shapes than that of the RBCs in adults. In addition, the hemoglobin (Hb) content in the cord RBCs from newborns was significantly higher. The Hb concentration in the cord RBCs was higher than that in the nonpregnant women or maternal RBCs, but they were within the physiological range of adults. Interestingly, the amplitudes of the dynamic membrane fluctuations in cord RBCs were comparable to those in nonpregnant women and maternal RBCs, suggesting that the deformability of cord RBCs is similar to that of healthy RBCs in adults.

Generation of Femtosecond Soliton Tweezers Using a Half-Panda System for Modeling the Trapping of a Human Red Blood Cell

Femtosecond optical tweezers are an effective tool for handling individual human red blood cells, as they allow for comparatively easy single cell manipulation with consequent diverse applications in biological science. Optical trapping at low power with short exposure time is desired in order to minimize damage in the vicinity of targeted tissue, and such a requirement can be achieved by using micro-ring resonators in an optical tweezers configuration. This paper describes a modified add/drop multiplexer system, known as half-panda ring resonator, which is suitable for human blood cells trapping and deployment during investigations on deformation and vibration behavior of biological cells. Dark-bright solitons and Gaussian beams are propagated within the proposed system, while the output features can be controlled via manipulation of specific parameters. This study details optical tweezers of FWHM of 33 and 100, and 90 and 152 femtoseconds that are generated at the drop and through ports of the system respectively

Quantitative phase imaging unit.

A simple and cost-effective method is presented for quantitative phase imaging. A common-path lateral phase shifting interferometer is realized through attaching a compact filter set to the output port of an existing microscope. The working principles, design criteria, and limitations are also derived and explained. In order to demonstrate the capability and applicability of the method, the optical phase images of a microsphere and individual human red blood cells are measured with high stability.

A Mathematical Framework for Combining Decisions of Multiple Experts toward Accurate and Remote Diagnosis of Malaria Using Tele-Microscopy

We propose a methodology for digitally fusing diagnostic decisions made by multiple medical experts in order to improve accuracy of diagnosis. Toward this goal, we report an experimental study involving nine experts, where each one was given more than 8,000 digital microscopic images of individual human red blood cells and asked to identify malaria infected cells. The results of this experiment reveal that even highly trained medical experts are not always self-consistent in their diagnostic decisions and that there exists a fair level of disagreement among experts, even for binary decisions (i.e., infected vs. uninfected). To tackle this general medical diagnosis problem, we propose a probabilistic algorithm to fuse the decisions made by trained medical experts to robustly achieve higher levels of accuracy when compared to individual experts making such decisions. By modelling the decisions of experts as a three component mixture model and solving for the underlying parameters using the Expectation Maximisation algorithm, we demonstrate the efficacy of our approach which significantly improves the overall diagnostic accuracy of malaria infected cells. Additionally, we present a mathematical framework for performing ‘slide-level’ diagnosis by using individual ‘cell-level’ diagnosis data, shedding more light on the statistical rules that should govern the routine practice in examination of e.g., thin blood smear samples. This framework could be generalized for various other tele-pathology needs, and can be used by trained experts within an efficient tele-medicine platform.

Light scattering of human red blood cells during metabolic remodeling of the membrane

We present the light scattering properties of individual human red blood cells (RBCs). We show that both the RBC static and dynamic scattering signals are altered by adenosine 5'-triphosphate (ATP)-driven membrane metabolic remodeling. To measure the light scattering signal from individual RBCs, we use diffraction phase microscopy together with a Fourier transform light scattering technique. RBC cytosolic ATPs are both chemically and metabolically depleted, and the corresponding scattering signals are compared with the light scattering signal of normal RBCs having physiologic levels of ATP.

Chemiluminescence resonance energy transfer-based detection for microchip electrophoresis.

Since the channels in micro- and nanofluidic devices are extremely small, a sensitive detection is required following microchip electrophoresis (MCE). This work describes a highly sensitive and yet universal detection scheme based on chemiluminescence resonance energy transfer (CRET) for MCE. It was found that an efficient CRET occurred between a luminol donor and a CdTe quantum dot (QD) acceptor in the luminol-NaBrO-QD system and that it was sensitively suppressed by the presence of certain organic compounds of biological interest including biogenic amines and thiols, amino acids, organic acids, and steroids. These findings allowed developing sensitive MCE-CL assays for the tested compounds. The proposed MCE-CL methods showed desired analytical figures of merit such as a wide concentration range of linear response. Detection limits obtained were approximately 10(-9) M for biogenic amines including dopamine and epinephrine and approximately 10(-8) M for biogenic thiols (e.g., glutathione and acetylcysteine), organic acids (i.e., ascorbic acid and uric acid), estrogens, and native amino acids. These were 10-1000 times more sensitive than those of previously reported MCE-based methods with chemiluminescence, electrochemical, or laser-induced fluorescence detection for quantifying corresponding compounds. To evaluate the applicability of the present MCE-CL method for analyzing real biological samples, it was used to determine amino acids in individual human red blood cells. Nine amino acids, including Lys, Ser, Ala, Glu, Trp, etc., were detected. The contents ranged from 3 to 31 amol/cell. The assay proved to be simple, quick, reproducible, and very sensitive.

Dynamic quantitative photothermal monitoring of cell death of individual human red blood cells upon glucose depletion

Red blood cells (RBCs) have been found to undergo "programmed cell death," or eryptosis, and understanding this process can provide more information about apoptosis of nucleated cells. Photothermal (PT) response, a label-free photothermal noninvasive technique, is proposed as a tool to monitor the cell death process of living human RBCs upon glucose depletion. Since the physiological status of the dying cells is highly sensitive to photothermal parameters (e.g., thermal diffusivity, absorption, etc.), we applied linear PT response to continuously monitor the death mechanism of RBC when depleted of glucose. The kinetics of the assay where the cell's PT response transforms from linear to nonlinear regime is reported. In addition, quantitative monitoring was performed by extracting the relevant photothermal parameters from the PT response. Twofold increases in thermal diffusivity and size reduction were found in the linear PT response during cell death. Our results reveal that photothermal parameters change earlier than phosphatidylserine externalization (used for fluorescent studies), allowing us to detect the initial stage of eryptosis in a quantitative manner. Hence, the proposed tool, in addition to detection of eryptosis earlier than fluorescence, could also reveal physiological status of the cells through quantitative photothermal parameter extraction.

Static and dynamic light scattering of healthy and malaria-parasite invaded red blood cells

We present the light scattering of individual Plasmodium falciparum-parasitized human red blood cells (Pf-RBCs), and demonstrate progressive alterations to the scattering signal arising from the development of malaria-inducing parasites. By selectively imaging the electric fields using quantitative phase microscopy and a Fourier transform light scattering technique, we calculate the light scattering maps of individual Pf-RBCs. We show that the onset and progression of pathological states of the Pf-RBCs can be clearly identified by the static scattering maps. Progressive changes to the biophysical properties of the Pf-RBC membrane are captured from dynamic light scattering.

Spectroscopic phase microscopy for quantifying hemoglobin concentrations in intact red blood cells

We report a practical method for label-free quantification of specific molecules using spectroscopic imaging of sample-induced phase shifts. Diffraction phase microscopy equipped with various wavelengths of light source is used to record wavelength-dependent phase images. We first perform dispersion measurements on pure solutions of single molecular species present in the cells, such as albumin and hemoglobin (Hb). With this prior calibration of molecular specific dispersion, we demonstrate the extraction of Hb concentration from individual human red blood cells. The end point of this study is noninvasive monitoring of physiological states of intact living cells.

One-dimensional jumping optical tweezers for optical stretching of bi-concave human red blood cells

We report the experimental demonstration of optical stretching of individual bio-concave human red blood cells (RBCs) with one-dimensional jumping optical tweezers. We trapped a RBC in isotonic buffer solution in a conventional stationary single-beam gradient-force optical trap and discretely scanned the trapping beam with an acousto-optic modulator such that the focal point of the trapping beam jumped back-and-forth between two fixed points. At the jumping frequency on the order of a 100 Hz and higher, and the jumping distance in the range of a few microns, the bi-concave RBC was stably trapped and stretched. The elongation of the stretched RBC was measured as a function of the beam-scanning amplitude, and the experimental results were explained qualitatively by a theoretical model.

Effect of temperature on the resistance of individual red blood cells to flow through capillary-sized apertures.

Low temperature can be expected to increase the resistance to deformation of red blood cells, but the effect of such changes on microcirculatory perfusion are unknown. We therefore analysed resistance to flow through capillary-sized apertures for individual human red blood cells, by micropipette aspiration (approximately 3 microm aperture) and pore transit analysis (approximately 5 microm), as well as average resistance to flow of red cell suspension through multipore filters (5-microm pores). Over a range decreasing from 37 to 0 degrees C, rates of flow of single cells through the 3- and 5-microm apertures decreased monotonically by 2.5- to 3-fold. The changes were similar in magnitude to that expected for the viscosity of aqueous fluid (2.5-fold increase). Average flow resistance measured by bulk filtration also increased in line with viscosity of water, while tendency to block pores was not increased. Micropipette aspiration of small membrane tongues showed that membrane rigidity increased as temperature was lowered, but by a factor rather less than the viscosity. Cell volume also responded rapidly to change in temperature, with lower temperature being associated with swelling, although this effect was much reduced in plasma compared with saline buffer. We conclude that, although increased resistance to deformation of red cells may impair microcirculation at low temperature, there is no structural change likely to induce more dramatic occlusion of flow. Moreover, the effect is comparable in magnitude to the increase predicted for changes in plasma and blood viscosity.

A study of the dynamic properties of the human red blood cell membrane using quasi-elastic light-scattering spectroscopy

A quasi-elastic light-scattering (QELS) microscope spectrometer was used to study the dynamic properties of the membrane/cytoskeleton of individual human red blood cells (RBCs). QELS is a spectroscopic technique that measures intensity fluctuations of laser light scattered from a sample. The intensity fluctuations were analyzed using power spectra and the intensity autocorrelation function, g(2)(tau), which was approximated with a single exponential. The value of the correlation time, Tcorr, was used for comparing results. Motion of the RBC membrane/cytoskeleton was previously identified as the source of the QELS signal from the RBC (R. B. Tishler and F. D. Carlson, 1987. Biophys. J. 51:993-997), and additional data supporting that conclusion are presented. Similar results were obtained from anucleate mammalian RBCs that have structures similar to that of the human RBC, but not for morphologically distinct, nucleated RBCs. The effect of altering the physical properties of the cytoplasm and the membrane/cytoskeleton was also studied. Osmotically increasing the cytoplasmic viscosity led to significant increases in Tcorr. Increasing the membrane cholesterol content and increasing the intracellular calcium content both led to decreased deformability of the human RBC. In both cases, the modified cells with decreased deformability showed an increase in Tcorr, demonstrating that QELS could measure biochemically induced changes of the membrane/cytoskeleton. Physiological changes were measured in studies of age-separated RBC populations which showed that Tcorr was increased in the older, less deformable cells.

Surface Elasticity and Viscosity of Red Cell Membrane

Force‐deformation curves are measured for individual human red blood cells by applying an equal and opposite force at diametrically opposite “points” on the red cell rim. Upon release of a cell, it rapidly recovers its unstressed disklike shape in approximately 0.3 sec. This deformation and subsequent recovery process is analyzed with a constitutive equation which describes the finite deformation at constant area of a two‐dimensional viscoelastic solid. From the analysis and the experimental results, values obtained for the “shear modulus of surface elasticity” and “coefficient of surface viscosity” are on the order of 4×10−3 dyn/cm and 5×10−4 dyn sec/cm (P cm), respectively.

Flow behavior of erythrocytes. II. Particle motions in concentrated suspensions of ghost cells

The detailed motions of individual human red blood cells (rbc) were studied under the microscope in transparent suspensions of red cell ghosts (serving as a model for whole blood) by tracking the particles in flow through tubes having radii R 0 of 32 to 80 μm at Reynolds numbers from 10 −3 to 0.3, and volume concentrations c from 0.10 to 0.93. The rbc velocity distributions were blunted from the parabolic at c > 0.2 with a region of plug flow of radius R c in the core of the tube where the velocities u M were measurably constant. R c increased with increasing c, and decreased with increasing flow rate Q and R 0. At c > 0.4, the rbc were continuously deformed from the resting biconcave shape, and their major axes became aligned within 20° of the flow. By contrast, gluteraldehyde-hardened red cells (hbc) continued to rotate with varying angular velocities, even at c = 0.93. The paths of the rbc exhibited erratic radial displacements whose magnitude and frequency at a given c increased with increasing shear rate, and in a given tube was greatest at 0.2 < c < 0.4 and with particles at radial positions 0.4 < R/ R 0 < 0.8. Appreciable radial displacements also occurred in the plug flow region where it was shown that shear rates of the order 0.01 u M/ R c existed. The distributions of rbc across the median plane of the tube were also measured. At high Q and c < 0.43 there was a pronounced movement of rbc to the periphery, perhaps due to differences in the rates of radial migration between rbc and ghosts stemming from differences in their densities.

Analysis of iron content in individual human red blood cells by electron microprobe and scanning electron microscope

The iron content of single human red blood cells has been assessed using electron microprobe analysis and scanning electron microscope. Cells for microanalysis consist of a fixed-washed and freeze-dried preparation. This preparative procedure improves stability and spatial resolution of the analytic method. The reference standard employs compressed pellets of purified iron-containing human hemoglobin and human albumin, respectively. The cell preparation and reference standard remain stable for long periods of time. Differences in Fe content and its distribution among individual red cells from both normal subjects and sickle cell anaemia patients have been measured by the quantitative and semi-quantitative techniques (wavelength and energy dispersive X-ray spectrometry). There are several points which can be made from this study. One concerns the estimate of iron on a cell by cell basis, the average variation among cells being slightly over 13% ranging from 6% to 19.4% by individuals (samples) or from 11% to 14% by ethnic groups. These differences are thus closely comparable across ethnic grouping with a possibility of high level in Asians and lower level in sickle cell anaemia patients.

Radioimmune assay for the Xg(a) surface antigen at the individual red cell level

An indirect radioimmune assay has been successfully used for the detection of the Xg(a) antigen on the surface of individual human red blood cells. The assay requires only microvolumes of reagents, is highly sensitive, and is equally reliable with fresh blood samples and with red blood cells stored in liquid nitrogen. The method has been used to estimate the number of Xg(a) binding sites. It may be useful to screen for quantitative isoalleles, as well as to investigate the alleged lack of participation of the Xg-locus in the inactivation of the X-chromosome.

The rate of sedimentation of individual human red blood cells.

AbstractThe sedimentation of individual cells was investigated in the context of the Stokes' equation for sedimenting spheres. Blood obtained from a finger prick was diluted 104 times by volume in Tris buffered Ringer solution (pH 7.4, 310 milliosmolar). A small sample of the suspension was put in a plastic, flat bottomed depression slide which was then closed with a coverslip. Thirty‐six runs were done in which a cell was kept in focus on an inverted microscope. The 250 μm descent was monitored on a two channel chart recorder. The orientation, determined by the observer as edge, oblique, or flat, and the verticle displacement, indicated by a ten turn potentiometer connected to the fine focussing knob, were recorded simultaneously. The cells studied indicated that cells are more frequently in the edge‐on orientation, that larger cells sediment faster than smaller cells despite the lower density for larger cells, and that cells wobble considerably. The biconcave cells made approximately three orientation changes per minute where one change in orientation was considered to be a rotation of 45° in any direction. Brownian motion is thought to be responsible for the wobble, or random rotations of the sedimenting cell. The edge‐to‐flat velocity ratio was 1.38 ± 0.30 S.D. which is significantly less than 1.5, the theoretical and experimental value for thin discs. There was a moderate dependence of sedimentation velocity on the square of the diameter (R = +0.5) in accordance with Stokes' Law. The variance in this relation can be accounted for by the known variation of cellular density within a normal population of red cells, and by the inherent errors of measurement.