title: Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy.
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.
authors.affiliation: University of Belgrade, Department of Chemical Engineering, Faculty of Technology and Metallurgy, Belgrade, Serbia.
authors.forename: Katarina
authors.initials: K
authors.lastname: Bukara
authors.affiliation: University of Belgrade, Institute of Physics Belgrade, Belgrade, Serbia.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Jovanic
authors.affiliation: University of Belgrade, Department of Chemical Engineering, Faculty of Technology and Metallurgy, Belgrade, SerbiacUniversity of Belgrade, Institute for Medical Research, Belgrade, Serbia.
authors.forename: Ivana T
authors.initials: IT
authors.lastname: Drvenica
authors.affiliation: University of Belgrade, Institute for Medical Research, Belgrade, Serbia.
authors.forename: Ana
authors.initials: A
authors.lastname: Stancic
authors.affiliation: University of Belgrade, Institute for Medical Research, Belgrade, Serbia.
authors.forename: Vesna
authors.initials: V
authors.lastname: Ilic
authors.affiliation: University of Belgrade, Institute of Physics Belgrade, Belgrade, Serbia.
authors.forename: Mihailo D
authors.initials: MD
authors.lastname: Rabasovic
authors.affiliation: University of Belgrade, Institute of Physics Belgrade, Belgrade, Serbia.
authors.forename: Dejan
authors.initials: D
authors.lastname: Pantelic
authors.affiliation: University of Belgrade, Institute of Physics Belgrade, Belgrade, Serbia.
authors.forename: Branislav
authors.initials: B
authors.lastname: Jelenkovic
authors.affiliation: University of Belgrade, Department of Chemical Engineering, Faculty of Technology and Metallurgy, Belgrade, Serbia.
authors.forename: Branko
authors.initials: B
authors.lastname: Bugarski
authors.affiliation: University of Belgrade, Institute of Physics Belgrade, Belgrade, SerbiadTexas A&M University at Qatar, Science Program, Doha, Qatar.
authors.forename: Aleksandar J
authors.initials: AJ
authors.lastname: Krmpot
mesh_terms: D000818:Animals; D004910:Erythrocyte Membrane / Q000737:chemistry; D004912:Erythrocytes / Q000737:chemistry* / Q000166:cytology*; D006454:Hemoglobins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D013552:Swine
chemical_list: D006454:Hemoglobins