Red Blood Cells Polarize Green Laser Light Revealing Hemoglobin′s Enhanced Non‐Fundamental Raman Modes

Abstract

In general, the first overtone modes produce weak bands that appear at approximately twice the wavenumber value of the fundamental transitions in vibrational spectra. Here, we report the existence of a series of enhanced non-fundamental bands in resonance Raman (RR) spectra recorded for hemoglobin (Hb) inside the highly concentrated heme environment of the red blood cell (RBC) by exciting with a 514.5 nm laser line. Such bands are most intense when detecting parallel-polarized light. The enhancement is explained through excitonic theory invoking a type C scattering mechanism and bands have been assigned to overtone and combination bands based on symmetry arguments and polarization measurements. By using malaria diagnosis as an example, we demonstrate that combining the non-fundamental and fundamental regions of the RR spectrum improves the sensitivity and diagnostic capability of the technique. The discovery will have considerable implications for the ongoing development of Raman spectroscopy for blood disease diagnoses and monitoring heme perturbation in response to environmental stimuli.

Visible Raman excitation laser induced power and exposure dependent effects in red blood cells

“…In the spin state marker region, the bands at 1547 cm −1 and 1604 cm –1 undergo an increase in intensity along with a concomitant decrease in the intensity of bands at 1585 cm −1 and 1636 cm –1 . These changes are known to be associated with transition of Fe from low‐spin to high‐spin state . Thus the power dependent spectral changes suggest a transition of Hb towards a high spin and +2 oxidation state of Fe which is a characteristic of deoxygenated Hb.…”

Label‐Free Molecular Imaging of Biological Cells and Tissues by Linear and Nonlinear Raman Spectroscopic Approaches

“…[145] Recently,t he binding between quinoline derivatives and ferriprotoporphyrin, as ac ore fragment before hemozoin formation, has been investigated in great detail. [149] In afew example studies,resonance Raman spectroscopy has also been applied to investigate tissues.Again, the context of high specificity for molecular species that fulfill the resonance conditions is of great advantage.R ecently,apilot study was conducted to monitor the level of tissue oxygenation by resonance Raman spectroscopy because evidence of hypoxia at the tissue level would provide very helpful information for an early recognition of hemorrhage.T he diagnosis as well as the localization is very critical, and fast, reliable tools for differentiation between shock and trauma are not available yet. Theresults of the study,w hich were supported by density functional calculations,s howed the formation of as alt bridge between the tertiary amino group of the quinoline and apropionate group of the heme,and different mechanisms of action for different quinoline derivatives.…”

“…Thea dditional information obtained by the detection of enhanced overtones improved the sensitivity and the diagnostic capability of the technique. [149] In afew example studies,resonance Raman spectroscopy has also been applied to investigate tissues.Again, the context of high specificity for molecular species that fulfill the resonance conditions is of great advantage.R ecently,apilot study was conducted to monitor the level of tissue oxygenation by resonance Raman spectroscopy because evidence of…”

“…Along with the investigations of red blood cells by resonance Raman spectroscopy, it was observed that red blood cells polarize green laser light, which can be utilized to enhance the non‐fundamental modes of hemoglobin. The additional information obtained by the detection of enhanced overtones improved the sensitivity and the diagnostic capability of the technique …”

Markerfreie molekulare Bildgebung biologischer Zellen und Gewebe durch lineare und nichtlineare Raman‐spektroskopische Ansätze

“…Diese Eigenschaft kann verwendet werden, um die nicht‐fundamentalen Schwingungsmoden des Hämoglobins zu verstärken. Die zusätzlichen Informationen aus der Detektion von verstärkten Obertonschwingungen erhöhten den diagnostischen Nutzen der Technik …”