Rudolf W. Kessler

Hyperspectral imaging: a review of best practice, performance and pitfalls for inline and online applications

The objective of this paper is to provide a comprehensive review of best practice in hyperspectral imaging. The paper starts to review the taxonomy of the different spectral imaging techniques together with their advantages and disadvantages. The appropriate selection of cameras and spectrographs and their figures of merit are discussed and a detailed description is given of how to qualify and calibrate a pushbroom imaging system for on-line and in-line control. Special emphasis is given to detection and avoidance of specular reflection which can severely distort quantification of the spectral response. Recommendations for an ideal Lambertian illumination are given and the effects of scatter and absorption are discussed when particulate systems are investigated. Here, first principles are introduced and strategies for how to separate scatter from absorption are developed. A simple method using the Kubelka and Munk approach is examined and separated scatter and pure absorption spectra are shown. The same procedure is applied to show the lateral distribution of the separated scatter and absorption properties of an active pharmaceutical ingredient embedded in an excipient. The terms penetration and information depth are discussed and an example of penetration depth profile over wavelengths is provided. Based on a good quality optical set-up and a validated measurement procedure, a practical procedure is described to analyse the data cube using the chemometrics toolbox for hyperspectral imaging. Finally, a survey on selected applications demonstrates the future potential of hyperspectral imaging.

Perspectives in process analysis

Process analysis is a transdisciplinary technology. Process chemists, process engineers, chemometricians, and many other technologists must work together and put more emphasis on the essentials of science of each discipline. This perspective analyzes potential strategies for intelligent production in the future. It emphasizes the need for a holistic approach where multimodality will be a bedrock supporting the production of smart materials in smart factories. Copyright

Quantitative Raman spectroscopy in turbid matter: reflection or transmission mode?

AbstractRaman intensities from reflection (XR) and transmission (XT) setups are compared by calculations based on random walk and analytical approaches with respect to sample thickness, absorption, and scattering. Experiments incorporating strongly scattering organic polymer layers and powder tablets of pharmaceutical ingredients validate the theoretical findings. For nonabsorbing layers, the Raman reflection and transmission intensities rise steadily with the layer thickness, starting for very thin layers with the ratio XT/XR = 1 and approaching for thick layers, a lower limit of XT/XR = 0.5. This result is completely different from the primary irradiation where the ratio of transmittance/reflectance decays hyperbolically with the layer thickness to zero. In absorbing materials, XR saturates at levels that depend strongly on the absorption and scattering coefficients. XT passes through a maximum and decreases then exponentially with increasing layer thickness to zero. From the calculated radial intensity spreads, it follows that quantitative transmission Raman spectroscopy requires diameters of the detected sample areas be about six times larger than the sample thickness. In stratified systems, Raman transmission allows deep probing even of small quantities in buried layers. In double layers, the information is independent from the side of the measurements. In triple layers simulating coated tablets, the information of XT originates mainly from the center of the bulk material whereas XR highlights the irradiated boundary region. However, if the stratified sample is measured in a Raman reflection setup in front of a white diffusely reflecting surface, it is possible to monitor the whole depth of a multiple scattering sample with equal statistical weight. This may be a favorable approach for inline Raman spectroscopy in process analytical technology. FigureRaman spectroscopy in turbid matter

Dark-field scattering microscopy for spectral characterization of polystyrene aggregates

Light scattering measurements of particle aggregates contain complex information which is difficult to decrypt. Dark-field scattering microscopy in the visible range is used to characterize multi-arranged polystyrene beads. First, measured light scattering spectra of single spheres are compared with the Mie theory. Then, additional spectral measurements of three different sample sets of sphere aggregates are carried out. The aggregates consist of homogeneous spheres and differ in number of spheres, arrangement and contact area. Principal component analysis is used to reduce the number of variables and achieve an accurate classification regarding the aggregate characteristics.

Structural properties of electrochemically designed porous oxide films on AlMg1

Abstract The characteristics of electrochemically formed porous oxide layers on aluminium alloys were investigated. The anodizing conditions were varied in a broad range (electrolyte: sulfuric acid, phosphoric acid and mixtures from these, concentration: 0.3–2.0 M, voltage: 5–10 V, time: 30–90 s, temperature: 25–50°C). The oxide layers were characterized via spectroscopic interference measurements to determine the oxide thickness. The morphology of this layer was also observed by AFM. The structural parameters of the oxide layer were characterized by electrochemical impedance spectroscopy (EIS). An equivalent circuit comprising two layers of barrier and porous layers was used to determine the characteristic parameters. The growth rate of oxide varied with electrolyte type, temperature and concentration. The proportionality constant of the barrier film thickness in relation to the applied voltage and the relative porosity of the film were calculated from this model. Three-dimensional response surfaces were constructed with the aid of an appropriate experimental design strategy which allows the study of not only the effects of the fabrication variables but also the interactions between these variables.

Penetration of Light into Multiple Scattering Media: Model Calculations and Reflectance Experiments. Part II: The Radial Transfer

In continuation of our contribution to “The Axial Transfer” (Appl. Spectr. 2012. 66(8): 934–943), this paper describes the distribution of localized incident radiation in multiple scattering layers of arbitrary thickness and analyzes the lateral intensity profiles of radiation leaving the sample from its illuminated and non-illuminated surfaces. The theoretical profiles are calculated with different approximations of the equation of transfer. We derive for both non-absorbing and absorbing layers simple analytical expressions and verify their accuracy and range of applicability by comparison with Monte Carlo simulations. Particular emphasis is given to the analysis of the radial absorption, an under-theorized and under-investigated feature that can help to identify weak or hidden absorbers. In addition, we contribute to the description of how the radial reflectance is affected by anisotropy or by error sources like multiple surface reflection for samples in glass cells or deflectance (sideway loss) of radiation in small samples. Finally, the theoretical results are compared with experimental data of radial reflectance for quasi non-absorbing and absorbing powder layers.

Multimodal spatially resolved near-field scattering and absorption spectroscopy

Far-field microscopy techniques are routinely used for the visualization of biological systems, but are limited according to Abbe`s criteria to about λ/2. The objective of this work is to integrate a solid immersion lens (SIL) as a near-field probe into a standard microscope spectrophotometer in order to perform polychromatic illumination near-field microscopy as well as near-field spectroscopy. The SIL concept can achieve a higher resolution than expected by the increase of the numerical aperture. Even with a tip diameter of 700nm and a tip point angle of 130° the lateral resolution is in the range of about 30 nm, therefore overcoming the tradeoff between the resolution and intensity restrictions in aperture limited SNOM probes. In this paper the optical setup of the system is described and some images of biological samples on a nanoscale with high contrast are presented.

Label-free multimodal microspectroscopic differentiation of glioblastoma tumor model cell lines combined with multivariate data analysis

Glioblastoma multiforme represents a highly lethal brain tumor. A tumor model has been developed based on the U-251 MG cell line from a human explant. The tumor model simulates different malignancies by controlled expression of the tumor suppressor proteins PTEN and TP53 within the cell lines derived from the wild type. The cells from each different malignant cell line are grown on slides, followed by a paraformaldehyde fixation. UV / VIS and IR spectra are recorded in the cell nuclei. For the differentiation of the cell lines a principal component analysis (PCA) is performed. The PCA demonstrates a good separation of the tumor model cell lines both with UV / VIS spectroscopy and with IR spectroscopy.

Spectroscopic imaging in the near field with an apertureless solid immersion lens microscope

To achieve high lateral resolution in microscopy, we exploit the localized electromagnetic field of a Solid Immersion Lens (SIL). The lens is mounted on a cantilever of a Scanning Probe Microscope (SPM) to allow a dynamic scan with constant tip-sample force. This unit can be integrated into a micro fluorescence (Zeiss UMSP) or Raman spectrometer (Renishaw) to allow spectroscopy and spectrally resolved imaging in the near field. Three methods can be applied with a lateral resolution of less than 30 nm: 1) Reflectance-SNOM: the sample is imaged by illuminating the surface through the SIL and detecting the reflected near-field. 2) Photon-tunneling-SNOM: the contrast is generated by the ability of the photons to tunnel through the energy barrier into the substrate. 3) Fluorescence- SNOM: the chromophore is excited and the fluorescence light is collected by the SIL. The collection efficiency for the fluorescence is increased by a factor of 10 due to the high refractive index of the SIL compared to conventional methods.

Laccase Catalyzed Indigo Carmine Transformation

SUMMARY A novel application of laccase for Indigo Carmine transformation with possible applications in enzymatic re-coloration of denim fabric is described. Indigo is used for dyeing denim fabric (“blue jeans”). For a fashionable “stone-washed” look, dyestuff is partially removed from dyed fabric mechanically with plim stones or enzymatically using cellulase-laccase cocktails. Laccases degrade indigo to isatin and anthranilic acid. However, upon systematic variation of pH, dye concentration, and enzyme activity we found that under appropriate reaction conditions a different reaction pathway leads to an intensively red coloured, stable reaction product of higher molecular weight. This reaction could be used to generate a wide variety of different new colour hues directly on the fabric.