Confocal Laser Scanning Microscope Imaging of Custom-Made Multi-Cylinder Phantoms: Theory and Experiment.

In this work, the image formation in a confocal laser scanning microscope (CLSM) is investigated for custom-made multi-cylinder phantoms. The cylinder structures were fabricated using 3D direct laser writing and consist of parallel cylinders with radii of 5 and 10 µm for the respective multi-cylinder phantom, with overall dimensions of about 200×200×200 µm3. Measurements were performed for different refractive index differences and by varying other parameters of the measurement system, such as pinhole size or numerical aperture (NA). For theoretical comparison, the confocal setup was implemented in an in-house developed tetrahedron-based and GPU-accelerated Monte Carlo (MC) software. The simulation results for a cylindrical single scatterer were first compared with the analytical solution of Maxwell's equations in two dimensions for prior validation. Subsequently, the more complex multi-cylinder structures were simulated using the MC software and compared with the experimental results. For the largest refractive index difference, i.e., air as the surrounding medium, the simulated and measured data show a high degree of agreement, with all the key features of the CLSM image being reproduced by the simulation. Even with a significant reduction in the refractive index difference by the use of immersion oil to values as low as 0.005, a good agreement between simulation and measurement was observed, particularly with respect to the increase in penetration depth.

Radiative transfer equation-based color prediction and color adjustment strategies.

This paper discusses different strategies for color prediction and matching. Although many groups use the two-flux model (i.e., the Kubelka-Munk theory or its extensions), we introduce a solution of the P N approximation for the radiative transfer equation (RTE) with modified Mark boundaries for the prediction of the transmittance and reflectance of turbid slabs with or without a glass layer on top. To demonstrate the capabilities of our solution, we have presented a way to prepare samples with different scatterers and absorbers where we can control and predict the optical properties and discussed three color-matching strategies: the approximation of the scattering and absorption coefficient, the adjustment of the reflectance, and the direct matching of the color valueL ∗ a ∗ b ∗.

Continuous Sizing and Identification of Microplastics in Water.

The pollution of the environment with microplastics in general, and in particular, the contamination of our drinking water and other food items, has increasingly become the focus of public attention in recent years. In order to better understand the entry pathways into the human food chain and thus prevent them if possible, a precise characterization of the particles concerning their size and material is indispensable. Particularly small plastic particles pose a special challenge since their material can only be determined by means of large experimental effort. In this work, we present a proof of principle experiment that allows the precise determination of the plastic type and the particle size in a single step. The experiment combines elastic light scattering (Mie scattering) with inelastic light scattering (Raman scattering), the latter being used to determine the plastic type. We conducted Monte Carlo simluations for the elastically scattered light for different kinds of plastics in a microfluidic cuvette which we could reproduce in the experiment. We were able to measure the Raman signals for different microplastics in the same measurement as the elastically scattered light and thereby determine their material. This information was used to select the appropriate Monte Carlo simulation data and to assign the correct particle size to different materials with only one calibration measurement.

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.

Application of Mie theory for enhanced size determination of microparticles using optical particle counters.

The approach to particle sizing with optical particle counters is often simple interpolation of calibration data. A method is presented that uses the results of Mie-theory-based simulations to describe the signal between calibration points, thus reducing the number of necessary calibration points or increasing the sizing accuracy significantly. Through the use of Mie theory, particles with a refractive index differing from the calibration particles can be measured without an individual calibration. The method can be used with custom research setups or commercially available optical particle counters with various detector designs. If needed, the method can be applied to particle counters for which only the light wavelength used is known. The method is tested using a commercially available optical particle counter with a polystyrene microsphere calibration, measuring polystyrene microspheres as well as THP-1 cells, Chinese hamster ovary cells, and yeast cells. Without material specific calibration, simple interpolation results in about half the actual particle sizes for these biological samples, whereas the presented method yields accurate results.

Method to determine the speckle characteristics of front projection screens.

We present a method to determine the speckle properties of front projection screens. Seven different screens are investigated in a backscattering geometry for 808 nm light. The speckle contrast reduction that results from polarization scrambling and reduced temporal coherence is modeled for the case of volume scattering in the screens. For this purpose, the screen's volume scattering path length distributions and depolarization characteristics are determined. This is done via a streak camera setup to measure the temporal broadening of ultrashort 50 fs light pulses scattered in the screens. We show that it is essential to properly select a projection screen with large volume roughness in order to achieve low speckle contrast values for moderate illumination bandwidths.