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.

Keywords: (020.4180) Multiphoton processes; (190.1900) Diagnostic applications of nonlinear optics.

Fig. 1

HbA as a negative control is imaged with low level fluorescence due to dissolved HbA molecules after deoxygenation by MPM excitation (A). Gelled HbS imaged by MPM after deoxygenation by the application of external heat (B). HbA does not form fibers upon deoxygenation. A line plot to the right of the images indicate the relative brightness along the white dotted line.

Fig. 2

Normalized TPEF spectra of gelled HbS. Excitation of TPEF at 800 nm and emission peak around 510 nm. Plotted spectra is the mean spectra of 3 separate micro-spectral measurements in different positions of the same sample and error bars indicate one standard deviation from the mean. Dashed line is included as guide to the eye.

Fig. 3

Time lapse images of HbS gel formation under different conditions. At low (<200 mg/ml) concentration (A-E), HbS slowly forms into nucleation clusters when photolysis by MPM excitation. At high (>250 mg/ml) concentrations (F-J), HbS fibers of more definite structure form more rapidly. Video content for series A-E available as 5033 kb in Visualization 1 and for series F-J available as 1501 kb in Visualization 2 as supplemental material.

Fig. 4

HbS fibers form with structure guided by surrounding structure or in the presence of magnetic fields. In a thin preparation (A) containing impurities, such as air bubbles, HbS fibers form around the impurities (B). In the presence of a magnetic field, the deoxygenated HbS fibers align perpendicularly to the applied field similar to the alignment of SCD erythrocytes elsewhere due to the diamagnetic nature of the molecule [1]. C and E diagram the orientation of the sample with respect to the horizontally and vertically applied fields respectively. D and F are the MPM images of fibers formed in the applied fields. Video content for B available as part of a 6540 kb Visualization 3, for D as a 1400 kb and for F as a 1501 Visualization 4 in supplemental material.