In this paper, we present a method to automatically quantify the three-dimensional (3D) volume of red blood cells (RBCs) using off-axis digital holographic microscopy. The RBCs digital holograms are recorded via a CCD camera using an off-axis interferometry setup. The RBCs' phase image is reconstructed from the recorded off-axis digital hologram by a computational reconstruction algorithm. The watershed segmentation algorithm is applied to the reconstructed phase image to remove background parts and obtain clear targets in the phase image with many single RBCs. After segmenting the reconstructed RBCs' phase image, all single RBCs are extracted, and the 3D volume of each single RBC is then measured with the surface area and the phase values of the corresponding RBC. In order to demonstrate the feasibility of the proposed method to automatically calculate the 3D volume of RBC, two typical shapes of RBCs, i.e., stomatocyte/discocyte, are tested via experiments. Statistical distributions of 3D volume for each class of RBC are generated by using our algorithm. Statistical hypothesis testing is conducted to investigate the difference between the statistical distributions for the two typical shapes of RBCs. Our experimental results illustrate that our study opens the possibility of automated quantitative analysis of 3D volume in various types of RBCs.

© 2012 Optical Society of Korea

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