3D morphometry of red blood cells by digital holography

Abstract

Three dimensional (3D) morphometric analysis of flowing and not-adherent cells is an important aspect for diagnostic purposes. However, diagnostics tools need to be quantitative, label-free and, as much as possible, accurate. Recently, a simple holographic approach, based on shape from silhouette algorithm, has been demonstrated for accurate calculation of cells biovolume and displaying their 3D shapes. Such approach has been adopted in combination with holographic optical tweezers and successfully applied to cells with convex shape. Nevertheless, unfortunately, the method fails in case of specimen with concave surfaces. Here, we propose an effective approach to achieve correct 3D shape measurement that can be extended in case of cells having concave surfaces, thus overcoming the limit of the previous technique. We prove the new procedure for healthy red blood cells (RBCs) (i.e., discocytes) having a concave surface in their central region.Comparative analysis of experimental results with a theoretical 3D geometrical model of RBC is discussed in order to evaluate accuracy of the proposed approach. Finally, we show that the method can be also useful to classify, in terms of morphology, different varieties of RBCs. V C 2014 International Society for Advancement of Cytometry Key terms red blood cells; morphological analysis; microfluidics; microscopy; digital holography; optical tweezers IN recent years, digital holography (DH) has gained credit as non-invasive and label-free diagnostic method in several research fields (1-6). Various microscope DH configurations have been proven to be powerful diagnostic tools for investigating micro-objects (7). For example, off-axis DH in microscopy is a very effective process for achieving high-precision, quantitative, phase-contrast mapping (QPM) of biological specimens as it allows measurements of optical thickness with nanometric accuracy (7). The main advantage of DH is the ability to furnish quantitative and intensity-independent microscope QPMs thus allowing quasi real-time and long time-lapse investigation of in vitro biological specimens (8-11). Nowadays, one of the main challenging goals in the field of bio-microscopy would be a diagnostic analysis through quantitative, label-free, and accurate 3D morphometric characterization. Several techniques have been proposed for the 3D morphological study of biological specimens, such as for example optical coherent tomography (12-16) and confocal microscopy (17-19), but they are not the most suitable methods in case of dynamic events such as flowing and not-adherent cells. Recently, a simple and fast method, based on shape from silhouette (SFS) algorithm, provided an effective solution for calculating cells biovolume and displaying their 3D shape (20). In general, 3D shape evaluation of an object is possible by observing a cell along different directions (21,22). In Ref. 20), the experimental arrangement was a combination of a DH microscope and optical tweezers (OT) (23). OT was employed for inducing selfrotation of the...

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