Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy

Katarina Bukara; Svetlana Z. Jovanić; Ivana T. Drvenica; Ana Stančić; Vesna Ilić; Mihailo D. Rabasović; Dejan V. Pantelić; Branislav M. Jelenković; Branko Bugarski; Aleksandar J. Krmpot

doi:10.1117/1.JBO.22.2.026003

9 February 2017 Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy

Katarina Bukara, Svetlana Z. Jovanić, Ivana T. Drvenica, Ana Stančić, Vesna Ilić, Mihailo D. Rabasović, Dejan V. Pantelić, Branislav M. Jelenković, Branko Bugarski, Aleksandar J. Krmpot

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Journal of Biomedical Optics, Vol. 22, Issue 2, 026003 (February 2017). https://doi.org/10.1117/1.JBO.22.2.026003

Abstract

The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells’ shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.

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Katarina Bukara, Svetlana Z. Jovanić, Ivana T. Drvenica, Ana Stančić, Vesna Ilić, Mihailo D. Rabasović, Dejan V. Pantelić, Branislav M. Jelenković, Branko Bugarski, and Aleksandar J. Krmpot "Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy," Journal of Biomedical Optics 22(2), 026003 (9 February 2017). https://doi.org/10.1117/1.JBO.22.2.026003

Received: 14 July 2016; Accepted: 24 January 2017; Published: 9 February 2017

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