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- MATERIALS AND METHODS
- LITERATURE CITED
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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. © 2008 International Society for Advancement of Cytometry
Mature erythrocytes (red blood cells, RBCs) represent the main cell type in circulating blood. They can be characterized by specific biconcave shape, high hemoglobin content, and absence of intracellular organelles such as the nucleus, mitochondria, or endoplasmic reticulum (1). Parameters such as RBC shape, volume, refractive index, and hemoglobin content are important characteristics that can be used as good indicators of the body's physiological state (2). For instance, erythrocyte volume distribution is altered in patients with anemia, folate and vitamin B12 deficiency, and microcytic anemia (2). The refractive properties of erythrocytes in diabetic patients differ significantly from those of healthy donors (3). Oxygen saturation also modulates the hemoglobin refractive index (4). Thus, monitoring the refractive index of erythrocytes can be used to assess their level of oxygen saturation. In addition, as the hemoglobin content is mainly responsible for the refractive index of the RBC, this parameter can be used as a measure of the mean cell hemoglobin concentration (MCHC) (5).
The first effort to measure the erythrocyte refractive index was conducted by perfusing RBCs with solutions of increasing refractive index until the cells exhibit no contrast under the phase-contrast microscope (6). Using this technique, a refractive index of 1.386 was determined for living erythrocytes (7). Since then a few attempts were made to assess the refractive index of normal RBCs using different approaches yielding values ranging from 1.367 to 1.410 (6, 8–11). To assess RBC volume, a few techniques have been implemented including light microscopy (12–14), impedance volume analysis (15), confocal fluorescence microscopy (16–19), light scattering (20, 21) as well as packed cell volume (PCV) calculation (22). These studies determined the volume of normal individual erythrocytes to be 80–120 fl, depending on the technique employed.
Recently, a new emerging imaging approach, namely quantitative phase microscopy (QPM) has been demonstrated to provide accurate 3D imaging of transparent living cells (11, 23–25). Although transparent specimens differ only slightly from their surrounding, in terms of optical properties, they have the capacity to induce wave front phase retardation on the transmitted wave. This natural phase retardation contrast, proportional to the thickness of the observed specimen, is a result of the difference in refractive indices between the specimen and the surrounding medium. Consequently, unlike traditional contrast-generating modes such as phase contrast (PhC), initially proposed by F. Zernike (26) or Nomarski's differential interference contrast (DIC) (27), QPM not only allows the visualization of transparent biological specimens but also provides quantitative information about both cell morphology and intracellular content related to the refractive index (25). The QPM technique we have developed, called digital holographic microscopy (DHM) is an interferometric approach based on the holographic principle (28). Briefly, from a single recorded hologram, quantitative phase images of living cells can be reconstructed by a numerical process (29). This numerical processing of holograms presents the great advantage of offering the means not only to reconstruct quantitative phase image but also to achieve a numerical compensation for aberration (30) and experimental noise (time drift, vibration, etc.). Consequently, although classical interferometric phase shift measurements are very sensitive to experimental noise (lens defects, vibrations, thermal drift, etc.), DHM allows to quantitatively measure phase shift corresponding to a fraction of the wavelength of the coherent light wave used, e.g., a few nanometers, with a high temporal stability and without using very demanding and costly opto-mechanical designs as required by conventional interferometric techniques. This explains why very few attempts to use conventional interferometric techniques have been reported in biology for real time living cell imaging. Nevertheless, interferometry was first used in 1957 by Barer (6) to measure refractive properties of RBC and later by Evans and Fung to measure erythrocyte dimensions (9).
As previously mentioned, the reconstructed quantitative phase image contains information about both the morphology and the refractive index of the monitored transparent specimen. As this dual information is intrinsically mixed, some strategies have been developed to separately assess the morphology and the refractive index. Kemper et al. (31) and Lue et al. (32) measured the cell integral refractive index by trapping cells between two cover glasses, whose distance apart is experimentally determined. On the other hand, a combined method has been proposed involving confocal microscopy to determine cell thickness and QPM to calculate the intracellular refractive index (33). We have developed a specific decoupling procedure, based on a concept initially proposed in (6, 9), allowing to directly calculate from the quantitative phase signal the corresponding cell morphology and integral refractive index. This procedure is particularly useful for measuring, under the same experimental conditions, both cell morphology changes and associated integral refractive index modifications occurring during biological processes (34).
In this article, we demonstrate the applicability of the DHM technique, in combination with a decoupling procedure, for the precise measurements of refractive indices and volumes of intact individual erythrocytes. The obtained values of erythrocyte mean cell volume (MCV), refractive index, and mean corpuscular hemoglobin (MCH) and concentration (MCHC) are compared with values obtained by an impedance volume analyzer, a laser scanning confocal microscope, and reference values found in the literature.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
- Supporting Information
DHM quantitatively measures optical phase shift, which provides quantitative information about both cell morphology and intracellular content related to the refractive index. Because of its high phase sensitivity and temporal stability, DHM enables the monitoring of fine dynamic processes in a living material with a great ease of use (25). In this study, we exploited such advantages to measure the refractive index of intact human RBC with a high accuracy.
Although the refractive index seems to be very sensitive to pathological alterations and thus to be important in diagnostics (3), only few attempts were previously made to assess this parameter. The situation may be explained by the complexity and inaccuracy of existing approaches. For example, the approach of Barer (6) involves an arbitrary judgment of the point when the erythrocytes sample and the solution of high concentration of albumin with n = 1.386 exhibit minimum contrast under the phase microscope. Others (9) directly measured the hemolyzed content of erythrocytes or the refractive index of packed cells. However, in both cases RBCs were far from their native state. Ghosh et al. (8) had found n = 1.405 and 1.410 for two normal volunteers by analyzing the angular dependence of laser light scattering from dense RBCs preparations. This approach uses the isovolumetrical sphering of RBCs due to difficulties in calculating light scattering from a biconcave discoid RBC. However, the procedure of sphering (40) itself can introduce artifacts. Defocusing microscopy (10) raised the value of n = 1.381 ± 0.005 for human RBC, but this technique requires exact knowledge of the RBC's height. Curl et al. (11) used a hypotonic agent to sphere the rat RBCs and measure their refractive index (n = 1.367) by QPM. This approach could underestimate the refractive index as the hypotonic shock causes a water entry into the cell. The value of n = 1.418, which we report in this work, is in the range of previously reported values but should be more precise as minimal RBC perturbation was employed and measurements were performed with high accuracy.
Another important RBC parameter, the cell volume, can be estimated as PCV-packed cell volume (22). In this approach, RBCs are subjected to prolonged 30 min centrifugation under relatively high centrifugal acceleration of 1,200–2,500g. To correct measurements for entrapped water, a radioactive label should be added for precise determinations (41). The volume of individual erythrocytes can be calculated by dividing the PCV by RBC count that generally yields a value of 90 fl. Such approach is laborious and time consuming, and alternative flow cytometry techniques are used now in the routine laboratory practice. One such flow cytometry technique is based on the detection of changes in electrical resistance produced by nonconductive particles such as cells suspended in an electrolyte when they pass through a narrow orifice (Coulter principle, (15)).
Another flow cytometry approach is to measure the amount of light scattered by individual red cells flowing through a narrow sensing aperture (20, 21); this technique allows for the independent measurement of the cell volume and mean hemoglobin concentration derived from the mean refractive index. On the other hand, one of the most sophisticated ways of estimating single cell volume is by confocal laser scanning microscopy (CLSM) (16–19). Indeed, the use of lipophilic fluorescent dyes to generate a specific cell membrane contrast or observation of autofluorescence and digital image processing of the image sets, consisting of serial optical sections across the cell, allows obtaining a 3D model of an individual cell. In particular, volume and shape estimation of living RBC have been obtained (42).
Figure 5 compares the results of volume measurements performed on the blood samples from the same healthy donor achieved by different techniques: Box plot distribution obtained with Sysmex KX-21 impedance volume analyzer (extrapolated from Fig. 4), CLSM and DHM, as well as some results from previous studies and the typical textbook value of 92 ± 9 fl (22) are indicated.
Figure 5. Box plot representation of the volume distribution obtained according to the method used. The box represents the median and the 25th and 75th percentile of the values, the whiskers represent the 10th and 90th percentile. DHM, digital holographic microscope (n = 36); confocal, Leica TCS-SP2 AOBS confocal laser scanning microscope (n = 34); KX-21, Sysmex KX-21 (6 measurements). References values from the literature are from (A) bright-field microscope (12) and (B) confocal microscope (42). (C) (22) is a textbook reference value.
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Figure 5 shows good agreement between results obtained by DHM and with the impedance volume analyzer. Results obtained by confocal microscopy are slightly higher, as results obtained in the literature with similar techniques such as light microscopy or CLSM. For example, Jay (12) using the approach described in (14), based on photographing individual RBCs hanging on edge, reports a typically MCV of 104.2. Difato et al. (42) obtained a value of 105 ± 5 fl for fixed RBCs by means of confocal microscopy and deconvolution procedures for calculations. The higher MCV values obtained with these techniques compared to DHM and impedance volume analyzer can be explained by the difficulty to precisely determine the cell edge and the need to use delicate deconvolution and analysis procedure (as described in this article and in (42)).
In Coulter sizing instruments, the magnitude of the resistive pulse generated by a cell depends not only on the cell volume but also on the cell shape. Rod-like particles traversing the instrument's orifice are thought to rise an electric pulse of “ideal” form, whereas signals obtained from spherical or disk-like particles need to be corrected by a shape factor (43). The shape dependence is even more complicated by the deformation the cells undergo while traversing the aperture (44). Thus, cytoplasmic viscosity (dependent on hemoglobin concentration in RBC) significantly influences cell deformation. Therefore, erythrocyte volume measurements are affected by hemoglobin concentrations, a quantity that varies from one cell to another (45). The parameters used for the measurement including dimensions of aperture and magnitude of electrical current also influence the shape of the volume distribution measured (46), for instance pulses from cells whose trajectories are close to the orifice can artificially broaden the volume distribution of the high volume side (as observed in Fig. 4). In practice, Coulter instruments are calibrated (47) by means of latex beads to obtain values for RBC mean cell volume close to that obtained by PCV. The same is true with another flow cytometry approach based on the light scattering by individual red cells flowing through a narrow sensing aperture. In this approach, the cells have to undergo an unphysiological isovolumetric spheric change before measurement (40). As PCV determination implies long and relatively strong centrifugation, the RBC can lose part of its water and the resulting MCV may be less than for intact unstressed RBC as suggested previously (13).
Concerning the hemoglobin content measurement, the MCHC results obtained with DHM (362 ± 7 g/l, mean ± standard error of the mean) are in good agreement with those obtained with the impedance volume analyzer (349 ± 12 g/l, mean ± accuracy given by the manufacturer). The Sysmex KX-21, which compares the optical density of lysed erythrocytes versus a calibration curve, allows to obtain a very precise measurement of the MCHC of cell populations and provides high throughput but is highly invasive as cell lysis is required. On the contrary, DHM measures the hemoglobin concentration of individual cells in a manner that is independent on the shape and state of the cell. The ability of DHM to measure the MCHC of erythrocytes independently and accurately may allow for the objective evaluation of the biological variation of this parameter in various RBC disorders such as anemia.
DHM is a new challenging interferometric imaging technique. In combination with the decoupling procedure, it allows to establish noninvasively the volume and intracellular refractive index of living cells with high accuracy. Specifically, the DHM measurements of erythrocyte volume and refractive index are in good agreement with data obtained by the more traditional techniques. Unlike flow cytometry, DHM does not currently exhibit high throughput capabilities but allows monitoring and measuring parameters (morphology, refractive index, MCHC) of individual erythrocytes. Further developments of the DHM technique, by applying automatic algorithms of analysis and increasing output capabilities, may promote it as the method that reveals subpopulations based on the parameters measured at a single cell level. On the other hand, DHM permits online tracking of changes in individual cells during, for example, osmotic fragility test or shear stress. It does not require utilization of fluorescent probes and employing of delicate and time-consuming deconvolution and image analysis procedures such as CLSM. Using DHM, the intracellular hemoglobin content of individual cells, a parameter altered in various pathological states, can be directly estimated from the phase measurements.