|Title:||Determination of optical and geometrical properties of blood cells and microparticle from light scattering measurements|
|Authors:||Gienger, Jonas Constantin, Physikalisch-Technische Bundesanstalt (PTB), Fachbereich 8.4, Mathematische Modellierung und Datenanalyse, ORCID: 0000-0003-1080-8874|
|Contributors:||HostingInstitution: Physikalisch-Technische Bundesanstalt (PTB), ISNI: 0000 0001 2186 1887|
|Resource Type:||Text / Dissertation|
|Publisher:||Physikalisch-Technische Bundesanstalt (PTB)|
|Rights:||Vervielfältigung nur zum eigenen persönlichen Gebrauch.|
|Relationships:||IsPartOf: ISSN 0341-6712
IsIdenticalTo: ISBN 978-3-95606-467-8
14.04 MB (14717124 Bytes)
MD5 Checksum: b6e6c9fd1a45995a023a11a763908d2e
SHA256 Checksum: 3a3e347f5268ba753648ea8c0ee1a3a22c1d775c8ccdaa48235f67d98e859f7d
|Keywords:||red blood cells ; refractive index ; light scattering ; microparticles ; flow cytometry|
|Abstract:||This thesis deals with the analysis of measurements of the scattering of light by red blood cells (RBCs) and artificial microparticles in order to determine their optical and geometrical properties. The light scattering properties of a particle or cell are determined by its shape and its complex refractive index (RI). RBCs have minimal internal structure and are a popular subject of fundamental research, but are also routinely examined with optical methods in laboratory medicine. Literature values for the real part of the RI (“real RI”) of RBCs and the oxygen-transport blood pigment hemoglobin (Hb) scatter widely, which hampers the quantitative analysis of light scattering data.
In this thesis, two complementary approaches are presented to determine the real RI of RBCs and Hb solutions: Firstly, the real RI in the near ultraviolet (UV), visible and near infrared region is determined from the well-known absorption spectrum of Hb solutions by Kramers-Kronig (KK) relations. To this end, the absorption spectrum is supplemented by a deep UV model for the peptide backbone of the metalloprotein Hb. This yields an accurate description of the dispersion features, but requires additional data for the real RI, e. g., from literature, to set the absolute scale. The second approach consists in an indirect, simultaneous determination of the size and RI of intact sphered RBCs in suspension from measurements of extinction spectra. These spectra describe how much light a particle or cell suspension removes from an incident beam due to scattering and absorption. They are analyzed by solving an inverse problem, where the direct problem consists in computing the average extinction cross section of a cell ensemble with known size distribution and optical properties using the Mie solution for light scattering by a sphere. The inverse problem is solved by a suitable few-parameter representation of the real RI and nonlinear optimization. After a demonstration of the method with synthetic polystyrene microbeads, it is applied to determine the Hb-concentration-specific increment of the real RI of oxygenated sphered RBCs for wavelengths between 290 nm and 1100 nm. The RI of other Hb variants can then be accurately determined in combination with the above-mentioned KK relations.
The RI data thus obtained are employed to assess the composition of artificial hemoglobin microparticles (HbMP), which might replace RBC concentrates in transfusion medicine. For the approval of clinical studies, characterization of their content of different Hb variants is required, in particular of oxygenated Hb, deoxygenated Hb and non-functional methemoglobin. This is achieved by a comparison between measured and computed extinction spectra for variable composition Lastly, a light scattering problem for RBCs in optical flow cytometry is considered. To interpret measured one- and two-dimensional histograms of the forward scattering cross section (FSC) of single native RBCs, the light scattering processes is numerically simulated with the discrete dipole approximation. A simple elongated RBC shape model is proposed and by comparison with measurement data, it is demonstrated that bimodal histograms FSC occur because of a combination of random orientation of the RBCs to the laser and deformation due to strong velocity gradients of the sheath flow in the flow cytometer.