- PhD Students
Using Multi-Photon Microscopy and Nonlinear optical phenomenon to observe signals in tissue and blood characteristic of sickle cell disease in order to develop in vivo monitoring which can be used to study, for example, the immunological and physiological characteristics of the disease.
Sickle cell disease (SCD), the most common genetic blood disorder, can cause a number of health complications including chronic pain, anemia, chronic infection and stroke. It is caused by a single point mutation resulting in an amino acid substitution in the oxygen transport molecule hemoglobin (Hb) which, upon deoxygenation, polymerizes into rigid fibers deforming red blood cells. These deformed, or sickled, red blood cells (RBC) have shortened circulation life, differing surface properties from oxygenated RBCs, are mechanically inflexible, and can cause clotting. Reliable DNA and biochemical assays are routinely performed for initial detection of the sickle cell trait and to quantify the severity of anemia. However, there is variation among the seriousness of symptoms and frequency of crisis states which are not well understood. Further, links between SCD and immune system dysfunction have been demonstrated leaving many unanswered questions about the mechanism and extent of immune dysregulation. It is of value to develop methods that allow for in vivo monitoring of the disease progression. This study proposes the monitoring of a number of intrinsic and optically detectable disease markers which may lead to useful methodologies for gaining insight into the physical factors responsible for symptoms of SCD.
We have proposed and have demonstrated ex vivo MPM imaging and identify endogenous distinctions between healthy and SCD tissue. These features can be mapped to physiological conditions and used to image the system over the course of disease progression to study the impact on the immune system. Further, it is proposed that a second harmonic signal can be generated from the sickle cell variant of deoxygenated hemoglobin, which has been shown to possess the required non-centrosymmetry for a non-zero, second order electric susceptibility tensor. The Second Harmonic Generation (SHG) could potentially be used to study sickled RBC circulation, especially in sensitive vasculature, e.g. brain where MPM related imaging has been successfully demonstrated elsewhere. The intrinsic Two-Photon Excited Fluorescence (TPEF) studied here is largely attributed to cellular NADH, characteristically blue-green (i.e. 460-520 nm), as well as Porphyrins and Lipofuscins, which are found in higher concentrations in cellular repair and inflammation machinery such as leukocytes and emit at longer wavelengths, (i.e. 500-660 nm).
Figure 1: 3D stacks of healthy mouse and SCD mouse splenic tissue. In the SCD tissue, the abundance of long-wave emission regions, which we identified spectrally as Iron-complex deposits, can easily be seen. Figure 2: Spectrum of several common biological intrinsic fluorophores compared with measured fluorescence of SCD tissue.
GD Vigil, AJ Adami, T Ahmed et al.; “Label-free and depth resolved optical sectioning of iron-complex deposits in sickle cell disease splenic tissue by multiphoton microscopy,” J. Biomed. Opt., 20(6), 066001 (2015). doi:10.1117/1.JBO.20.6.066001.