Three-dimensional volumetric measurement of red blood cell motion using digital holographic microscopy

Measurement of blood flow with high spatial and temporal resolutions in a three-dimensional (3D) volume is a challenge in biomedical research fields. In this study, digital holographic microscopy is used to measure the 3D motion of human red blood cells (RBCs) in a microscale volume. The cinematographic holography technique, which uses a high-speed camera, enabled the continuous tracking of individual RBCs in a microtube flow. Several autofocus functions that quantify the sharpness of reconstructed RBC images are evaluated to locate the accurate depthwise position of RBCs. In this study, the squared Laplacian function yields the smallest depth of focus and locates the depthwise positions of RBCs with a root mean square error of 2.3 microm. By applying this method, we demonstrate the measurement of four-dimensional (space and time) trajectories as well as 3D velocity profiles of RBCs. The measurement uncertainties of the present method are also discussed.