Domes and semi-capsules as model systems for infrared microspectroscopy of biological cells.

It is well known that infrared microscopy of micrometer sized samples suffers from strong scattering distortions, attributed to Mie scattering. The state-of-the-art preprocessing technique for modelling and removing Mie scattering features from infrared absorbance spectra of biological samples is built on a meta model for perfect spheres. However, non-spherical cell shapes are the norm rather than the exception, and it is therefore highly relevant to evaluate the validity of this preprocessing technique for deformed spherical systems. Addressing these cases, we investigate both numerically and experimentally the absorbance spectra of 3D-printed individual domes, rows of up to five domes, two domes with varying distance, and semi-capsules of varying lengths as model systems of deformed individual cells and small cell clusters. We find that coupling effects between individual domes are small, corroborating previous related literature results for spheres. Further, we point out and illustrate with examples that, while optical reciprocity guarantees the same extinction efficiency for top vs. bottom illumination, a scatterer's internal field may be vastly different in these two situations. Finally, we demonstrate that the ME-EMSC model for preprocessing infrared spectra from spherical biological systems is valid also for deformed spherical systems.

Effects of the coupling of dielectric spherical particles on signatures in infrared microspectroscopy.

Infrared microspectroscopy is a powerful tool in the analysis of biological samples. However, strong electromagnetic scattering may occur since the wavelength of the incident radiation and the samples may be of comparable size. Based on the Mie theory of single spheres, correction algorithms have been developed to retrieve pure absorbance spectra. Studies of the scattering characteristics of samples of different types, obtained by microspectroscopy, have been performed. However, the detailed, microscopic effects of the coupling of the samples on signatures in spectra, obtained by infrared microspectroscopy, are still not clear. The aim of this paper is to investigate how the coupling of spherical samples influences the spectra. Applying the surface integral equation (SIE) method, we simulate small dielectric spheres, arranged as double-spheres or small arrays of spheres. We find that the coupling of the spheres hardly influences the broad oscillations observed in infrared spectra (the Mie wiggles) unless the radii of the spheres are different or the angle between the direction of the incident radiation and the normal of the plane where the spheres are located is large. Sharp resonance features in the spectra (the Mie ripples) are affected by the coupling of the spheres and this effect depends on the polarization of the incident wave. Experiments are performed to verify our conclusions.

The effect of deformation of absorbing scatterers on Mie-type signatures in infrared microspectroscopy.

Mie-type scattering features such as ripples (i.e., sharp shape-resonance peaks) and wiggles (i.e., broad oscillations), are frequently-observed scattering phenomena in infrared microspectroscopy of cells and tissues. They appear in general when the wavelength of electromagnetic radiation is of the same order as the size of the scatterer. By use of approximations to the Mie solutions for spheres, iterative algorithms have been developed to retrieve pure absorbance spectra. However, the question remains to what extent the Mie solutions, and approximations thereof, describe the extinction efficiency in practical situations where the shapes of scatterers deviate considerably from spheres. The aim of the current study is to investigate how deviations from a spherical scatterer can change the extinction properties of the scatterer in the context of chaos in wave systems. For this purpose, we investigate a chaotic scatterer and compare it with an elliptically shaped scatterer, which exhibits only regular scattering. We find that chaotic scattering has an accelerating effect on the disappearance of Mie ripples. We further show that the presence of absorption and the high numerical aperture of infrared microscopes does not explain the absence of ripples in most measurements of biological samples.