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 setup 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.
The article presents two general equations of radiation penetration into layers of diffuse reflectors. One of the equations describes the depth origins of reflection, the other the depth profiles of absorption. The equations are evaluated within the theory of radiative transfer applying various degrees of analytical approximations and Monte Carlo simulations. The data are presented for different scattering and absorption coefficients, arbitrary layer thicknesses, collimated and diffused irradiation, and anisotropic forward scattering. The calculated mean depths of reflection are always lower than the mean depths of absorption. For nearly non-absorbing layers, the mean depths of absorption are about one third of the physical layer thickness. In contrast, penetration saturates for strong absorbers at very low depth levels. From the simulated data, methods are derived for the determination of the penetration depth from reflectance and transmittance data of thin layers or from radially diffused reflectance profiles upon spot irradiation. The methods are experimentally verified for a series of metal oxide powders with particle sizes ranging from much smaller to much larger than the wavelength of irradiation and for microcrystalline cellulose stained with different concentrations of an organic dye.
A new two‐dimensional fluorescence sensor system was developed for in‐line monitoring of mammalian cell cultures. Fluorescence spectroscopy allows for the detection and quantification of naturally occurring intra‐ and extracellular fluorophores in the cell broth. The fluorescence signals correlate to the cells’ current redox state and other relevant process parameters. Cell culture pretests with twelve different excitation wavelengths showed that only three wavelengths account for a vast majority of spectral variation. Accordingly, the newly developed device utilizes three high‐power LEDs as excitation sources in combination with a back‐thinned CCD‐spectrometer for fluorescence detection. This setup was first tested in a lab design of experiments study with process relevant fluorophores proving its suitability for cell culture monitoring with LOD in the μg/L range. The sensor was then integrated into a CHO‐K1 cell culture process. The acquired fluorescence spectra of several batches were evaluated using multivariate methods. The resulting batch evolution models were challenged in deviating and “golden batch” validation runs. These first tests showed that the new sensor can trace the cells’ metabolic state in a fast and reliable manner. Cellular distress is quickly detected as a deviation from the “golden batch”.
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