We have utilized the two photon excitation fluorescence (TPEF) microscopy to map the spatial distribution of residual hemoglobin. The home-made experimental set up for NLSM utilizes the train of the femtosecond pulses from Ti:Sapphire laser at 730nm, repetition rate 76MHz, and pulse duration 160fs. Porcine slaughterhouse blood and human outdated blood were used as a starting biological material. The erythrocyte ghosts were prepared by gradual hypotonic hemolysis [3]. First we used TPEF microscopy to image the intact erythrocytes at single cell level and to study their morphologies, discocyte of human and echynocite of porcine erythrocytes. We have shown that the distribution of hemoglobin in intact erythrocytes follows the cells’ shape with pronounced abundance in the proximity of cell membrane [4]. The TPEF images have also revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, residual hemoglobin localized on intracellular side of the ghost membranes [4]. The TPEF estimated hemoglobin distribution in intact erythrocytes and residual hemoglobin distribution in erythrocyte ghosts could be of importance in biotechnological processes but also in diagnosis of different pathological conditions.

[1] W. Zheng et al, Biomed Opt Express 2, 71-79 (2010) [2] V. Leuzzi, et al, J. Inherit. Metab. Dis., 1-12 (2016) [3] I. Kostić, wt al, Colloids Surf B 122, 250-259 (2014) [4] K. Bukara et al, J. Biomed. Opt. 22, 26003 (2017)