Research Papers: Imaging

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

[+] Author Affiliations
Katarina Bukara, Branko Bugarski

University of Belgrade, Department of Chemical Engineering, Faculty of Technology and Metallurgy, Belgrade, Serbia

Svetlana Jovanić, Mihailo D. Rabasović, Dejan Pantelić, Branislav Jelenković

University of Belgrade, Institute of Physics Belgrade, Belgrade, Serbia

Ivana T. Drvenica

University of Belgrade, Department of Chemical Engineering, Faculty of Technology and Metallurgy, Belgrade, Serbia

University of Belgrade, Institute for Medical Research, Belgrade, Serbia

Ana Stančić, Vesna Ilić

University of Belgrade, Institute for Medical Research, Belgrade, Serbia

Aleksandar J. Krmpot

University of Belgrade, Institute of Physics Belgrade, Belgrade, Serbia

Texas A&M University at Qatar, Science Program, Doha, Qatar

J. Biomed. Opt. 22(2), 026003 (Feb 09, 2017). doi:10.1117/1.JBO.22.2.026003
History: Received July 14, 2016; Accepted January 24, 2017
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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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© 2017 Society of Photo-Optical Instrumentation Engineers

Citation

Katarina Bukara ; Svetlana Jovanić ; Ivana T. Drvenica ; Ana Stančić ; Vesna Ilić, et al.
"Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy", J. Biomed. Opt. 22(2), 026003 (Feb 09, 2017). ; http://dx.doi.org/10.1117/1.JBO.22.2.026003


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