Erythrocyte volumetric measurements in imaging flow cytometry using simultaneous three-wavelength digital holographic microscopy.

We report a cross-talk free simultaneous three-wavelength digital holographic microscopy setup for spectroscopic imaging of biological cells during flow. The feasibility of the proposed measurement technique is demonstrated on erythrocytes, due to their unique morphology and dependency of hemoglobin (Hb) molecule absorption on wavelength. From the spectroscopic quantitative phase profiles of cells acquired during flow in a microfluidic device, we decoupled the refractive index and the physical thickness. We then used our quantitative phase imaging results to dynamically calculate the mean cell volume (MCV), mean corpuscular Hb concentration (MCHC), mean corpuscular Hb content (MCH) and sphericity index.