Dynamic three-wavelength imaging and volumetry of flowing cells with doubled field of view by six-pack holography

We present a new six-pack holography (6PH) system for simultaneous acquisition of six off-axis holograms in a single exposure from two fields of view and three wavelengths. The system is based on an external 6PH module, placed at the output of an inverted microscope, presenting a significantly more elegant solution compared to previous 6PH systems, which were based on cumbersome Mach–Zehnder interferometers built around the sample. This is also the first time in which six-pack holography is utilized for multi-wavelength imaging. We demonstrate using the new system to acquire volumetric data of flowing erythrocytes with twice the field of view of previous multi-wavelength holographic techniques. This volumetric data includes mean corpuscular volume, mean corpuscular hemoglobin (Hb) concentration, mean corpuscular Hb, and sphericity.