Detection of Calcium-induced morphological changes on RBCs by digital holographic microscopy and blinking optical tweezers

Ca+2 level in the circulating red blood cells (RBCs) takes part not only in controlling biophysical properties, but also affects the membrane composition, and its morphological and rheological properties. Excessive accumulation of Ca2+ within the cells is associated with a number of important pathological diseases. In this paper, by the use of digital holographic microscopy (DHM), we quantitatively analyzed the volumetric behavior of RBC membrane under influence of excess Calcium ions. DHM in a transmission mode is an effective tool for quantitative visualization of phase objects. By deriving the associated phase changes 3D information on the morphology variation of the cells at arbitrary time scales is obtained. Individual cells are immobilized by the use of optical tweezers and are monitored live with DHM system, while the concentration of Ca2+ ions in the buffer is changed simultaneously. We utilized blinking optical tweezers, by inserting an optical chopper to modulate intensity of the trapping laser beam. Blinking optical tweezers, while keeping the cell trapped during the experiments, ensures of minimizing the photo-damage of trapping laser beam on the cell. Our experimental results are in agreement with previous biological studies and predictions, and experimental observations of living RBCs under Ca2+ influence.