The real part of the refractive index (RI) of aqueous solutions of human hemoglobin is computed from their absorption spectra in the wavelength range 250 nm-1100 nm using the Kramers-Kronig (KK) relations and the corresponding uncertainty analysis is provided. The strong ultraviolet (UV) and infrared absorbance of the water outside this spectral range were taken into account in a previous study employing KK relations. We improve these results by including the concentration dependence of the water absorbance as well as by modeling the deep UV absorbance of hemoglobin's peptide backbone. The two free parameters of the model for the deep UV absorbance are fixed by a global fit.

Control over nanoscale patterning of ultrathin molecular films plays an important role both in natural as well as artificial nanosystems. Here we report on nanophase separated patterns of water and ethanol within monomolecularly thin films confined between the cleavage plane of mica and single or a few layers of graphene. Employing scanning force microscopy of the graphene layers conforming to the molecular films we quantify the patterns using the ethanol-water cross correlation and the autocorrelation of domain wall directions. They reveal that lateral pattern dimensions grow and the domain walls stiffen upon increasing the thickness of the graphene multilayers. We attribute the control of the patterns through the graphene layers to the competition between the mechanical deformation energy of the graphene sheets and the electrostatic repulsion of dipoles normal to the interface. The latter results from charge transfer between graphene and the molecules confined between mica and graphene.

The knowledge of optical properties of biological cells is essential to interpret their interaction with light and to derive morphological information and parameters associated with cell function like the oxygen transport capacity of human red blood cells (RBCs). We present a method to determine the dependence between the refractive index (RI) of human RBCs and their intracellular hemoglobin (Hb) concentration from spectral extinction measurements of a cell suspension. The procedure is based on the analysis of the corresponding ensemble averaged extinction cross section . Thus far two complementary approaches have been taken to derive RIs of RBCs. The first one uses homogeneous macroscopic samples prepared by hemolysis for the destruction of the RBCs’ membranes and subsequent centrifugation. A second approach is the determination of RIs of single intact cells by microscopic investigation. These techniques are limited to a few discrete wavelengths or a rather narrow wavelength range. In addition most of these techniques require additional information about the concentration dependence. In contrast, our approach yields the RI increment with Hb concentration of intact, reversibly isovolumetrically sphered, oxygenated RBCs over a wide wavelength range from 290 nm to 1100 nm from macroscopic measurements.

A method is presented to infer simultaneously the wavelength-dependent real refractive index (RI) of the material of microspheres and their size distribution from extinction measurements of particle suspensions. To derive the averaged spectral optical extinction cross section of the microspheres from such ensemble measurements, we determined the particle concentration by flow cytometry to an accuracy of typically 2% and adjusted the particle concentration to ensure that perturbations due to multiple scattering are negligible. For analysis of the extinction spectra, we employ Mie theory, a series-expansion representation of the refractive index and nonlinear numerical optimization. In contrast to other approaches, our method offers the advantage to simultaneously determine size, size distribution, and spectral refractive index of ensembles of microparticles including uncertainty estimation.

Light scattering by single cells is widely applied for flow cytometric differentiation of cells. However, even for human red blood cells (RBCs), which can be modeled as homogeneous dielectric particles, the potential of light scattering is not yet fully exploited. We developed a dedicated flow cytometer to simultaneously observe the forward scattering cross section (FSC) of RBCs for orthogonal laser beams with incident wave vectors ì k 1 and ì k 2 . At a wavelength λ = 632.8 nm, bimodal distributions are observed in two-dimensional dot plots of FSC( ì k 1 ) vs. FSC( ì k 2 ), which result from the RBCs' random orientation around the direction of flow, as well as from the distributions of their size and their optical properties. Typically, signals of 7.5 × 10 4 RBCs were analyzed. We actively oriented the cells in the cytometer to prove that orientation is the main cause of bimodality. In addition, we studied the wavelength dependence of FSC( ì k 1 ) using λ = 413.1 nm, 457.9 nm, 488 nm and 632.8 nm, covering both weak and strong light absorption by the RBCs. Simulations of the light scattering by single RBCs were performed using the discrete dipole approximation (DDA) for a range of sizes, orientations and optical properties to obtain FSC distributions from RBC ensembles. Using the axisymmetric biconcave equilibrium shape of native RBCs, the experimentally observed distributions cannot be reproduced. If, however, an elongated shape model is employed that accounts for the stretching of the cell by hydrodynamic forces in the cytometer, the features of the strongly bimodal measured frequency distributions are reproduced by the simulation. Elongation ratios significantly greater than 1 in the range of 1.5 to 2.5 yield the best agreement between experiments and simulated data.