Photophysical characterization of sickle cell disease hemoglobin by multi-photon microscopy

Abstract

The photophysical properties of human sickle cell disease (SCD) Hemoglobin (Hb) is characterized by multi-photon microscopy (MPM). The intrinsic two-photon excited fluorescence (TPEF) signal associated with extracted hemoglobin was investigated and the solidified SCD variant (HbS) was found to demonstrate broad emission peaking around 510 nm when excited at 800 nm. MPM is used to dynamically induce and image HbS gelling by photolysis of deoxygenated HbS. For comparison, photolysis conditions were applied to a healthy variant of human hemoglobin (HbA) and found to remain in solution not forming fibers. The use of this signal to study the mechanism of HbS polymerization associated with the sickling of SCD erythrocytes is discussed.

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

“…Until now it was described that isolated sickle cell disease hemoglobin (HbS) and malaria-infected red blood cells could be analyzed and photophysically characterized by multiphoton microscopy. 28,38 The overall presence of residual hemoglobin in the final suspensions of erythrocyte ghosts was primarily determined by cyanmethemoglobin assay (Fig. 5).…”

“…26 In addition to demonstration of the imaging possibilities of hemoglobin by the aforementioned techniques, 2PE has recently been applied in examination of particular biomedical problems related to erythrocytes: tracking blood vessels within the tissues 27 and photophysical characterization of sickle cell disease hemoglobin. 28 In this work, we report label-free imaging of two kinds of erythrocytes, porcine and outdated human ones, at single cell level by 2PE microscopy, with the specific aim of investigating the spatial distribution (i.e., for mapping) of hemoglobin within intact erythrocytes and residual hemoglobin in erythrocyte membranes obtained after a gradual hypotonic hemolysis process. Furthermore, the quantity of residual hemoglobin was determined by analysis of 2PE images relative to the hemoglobin concentration in intact erythrocytes.…”

Introduction to the bio-optics: design and application

“…In this feature issue of Biomedical Optics Express, we have collected several papers that represent some of the technologies discussed at the Bio-Optics: Design and Application. These papers highlight advances in diagnostic microscopy [1][2][3][4][5], optical coherence tomography [6], multimodal imaging [7,8] and selected applications [9,10]. Together, they present a set of interesting instrumentation developments in the field biomedical optics.…”