Generic and Model-Based Calibration Method for Spatial Frequency Domain Imaging with Parameterized Frequency and Intensity Correction.

Spatial frequency domain imaging (SFDI) is well established in biology and medicine for non-contact, wide-field imaging of optical properties and 3D topography. Especially for turbid media with displaced, tilted or irregularly shaped surfaces, the reliable quantitative measurement of diffuse reflectance requires efficient calibration and correction methods. In this work, we present the implementation of a generic and hardware independent calibration routine for SFDI setups based on the so-called pinhole camera model for both projection and detection. Using a two-step geometric and intensity calibration, we obtain an imaging model that efficiently and accurately determines 3D topography and diffuse reflectance for subsequently measured samples, taking into account their relative distance and orientation to the camera and projector, as well as the distortions of the optical system. Derived correction procedures for position- and orientation-dependent changes in spatial frequency and intensity allow the determination of the effective scattering coefficient µs' and the absorption coefficient µa when measuring a spherical optical phantom at three different measurement positions and at nine wavelengths with an average error of 5% and 12%, respectively. Model-based calibration allows the characterization of the imaging properties of the entire SFDI system without prior knowledge, enabling the future development of a digital twin for synthetic data generation or more robust evaluation methods.

Imaging of custom-made single scatterers with the confocal laser scanning microscope.

In this work, we investigate image formation in the confocal laser scanning microscope for different single scatterers, both theoretically and experimentally. For spherical scatterers, an effective and fast algorithm was implemented to calculate the confocal image for different diameters and wavelengths. Measurements on a polystyrene sphere (PS) with a diameter of 20 µm confirmed the expected effects, for example, the appearance of a central signal similar to the point spread function of the optical system. Custom single scatterers were produced using 3D-direct laser writing (DLW), including a sphere with dimensions comparable to the aforementioned PS sphere. Despite an inevitably lower surface quality and symmetry, only minor differences were observed in the confocal image of the 3D-DLW sphere compared to a near-perfect PS sphere. Having verified the experimental images of spheres with the computed theoretical data, confocal measurements of four platonic bodies produced by 3D-DLW were measured with the goal to contribute to the understanding of image formation involving more complex scattering geometries.

Chlorophyll- and anthocyanin-rich cell organelles affect light scattering in apple skin.

Apple skin contains several groups of strongly absorbing cell organelles with pigments that change dynamically in type and concentration during fruit maturation. Chlorophylls and carotenoids, both primarily involved in photosynthesis, are found in the grana of chloroplasts, while anthocyanin vacuolar inclusions (AVIs) accumulate for light protection in red-skinned cultivars. A Mie model describing light scattering by absorbing spherical particles in a non-absorbing medium allowed to theoretically investigate the explicit influence of grana and AVIs on the effective scattering coefficient [Formula: see text] and the absorption coefficient [Formula: see text]. The reconstruction of the complex refractive indices of the organelles predicted anomalous dispersion, i.e., a local increase in the real part of the refractive index in the spectral regions with high chlorophyll and anthocyanin absorption, in agreement with the Kramers-Kronig relations. As a result, peaks in [Formula: see text] were predicted to be shifted to longer wavelengths compared to the corresponding [Formula: see text] bands. This selective scattering effect was confirmed experimentally with integrating sphere measurements for red- or green-skinned apple samples of the cultivars 'Elstar', 'Gala' or 'Jonagold'. Comparison between simulations and measurements indicated that the Soret bands of chlorophyll a and chlorophyll b are at 435 nm and 469 nm, respectively, and overlap with the absorption of carotenoids, whose red-most edge is at 488 nm. For anthocyanin absorption, a pronounced blue shift from 550 to 520 nm was observed, indicating structural or chemical changes of AVIs.