Thomas Beuermann

Multimodal imaging gains increasing popularity for biomedical applications. This article presents the design of a novel multimodal imaging system. The centerpiece is a light microscope operating in the incident and transmitted light mode. Additionally, Raman spectroscopy and VIS/NIR reflectance spectroscopy are adapted. The proof-of-concept is realized to distinguish between grey matter (GM) and white matter (WM) of normal mouse brain tissue. Besides Raman and VIS/NIR spectroscopy, the following optical microscopy techniques are applied in the incident light mode: brightfield, darkfield, and polarization microscopy. To complement the study, brightfield images of a hematoxylin and eosin (H&E) stained cryosection in the transmitted light mode are recorded using the same imaging system. Data acquisition based on polarization microscopy and Raman spectroscopy gives the best results regarding the tissue differentiation of the unstained section. In addition to the discrimination of GM and WM, both modalities are suited to highlight differences in the density of myelinated axons. For Raman spectroscopy, this is achieved by calculating the sum of two intensity peak ratios (I₂₈₅₇ + I₂₈₈₈)/I₂₉₃₀ in the high-wavenumber region. For an optimum combination of the modalities, it is recommended to apply the molecule-specific but time-consuming Raman spectroscopy to smaller regions of interest, which have previously been identified by the microscopic modes.

Raman and mid-infrared (MIR) spectroscopy are useful tools for the specific detection of molecules, since both methods are based on the excitation of fundamental vibration modes. In this study, Raman and MIR spectroscopy were applied simultaneously during aerobic yeast fermentations of Saccharomyces cerevisiae. Based on the recorded Raman intensities and MIR absorption spectra, respectively, temporal concentration courses of glucose, ethanol, and biomass were determined. The chemometric methods used to evaluate the analyte concentrations were partial least squares (PLS) regression and multiple linear regression (MLR). In view of potential photometric sensors, MLR models based on two (2D) and four (4D) analyte-specific optical channels were developed. All chemometric models were tested to predict glucose concentrations between 0 and 30 g L⁻¹, ethanol concentrations between 0 and 10 g L⁻¹, and biomass concentrations up to 15 g L⁻¹ in real time during diauxic growth. Root-mean-squared errors of prediction (RMSEP) of 0.68 g L⁻¹, 0.48 g L⁻¹, and 0.37 g L⁻¹ for glucose, ethanol, and biomass were achieved using the MIR setup combined with a PLS model. In the case of Raman spectroscopy, the corresponding RMSEP values were 0.92 g L⁻¹, 0.39 g L⁻¹, and 0.29 g L⁻¹. Nevertheless, the simple 4D MLR models could reach the performance of the more complex PLS evaluation. Consequently, the replacement of spectrometer setups by four-channel sensors were discussed. Moreover, the advantages and disadvantages of Raman and MIR setups are demonstrated with regard to process implementation.

Fluorescence emission intensity is an important indicator for characterising tissue. This study extends our previous work and presents an algorithm to determine the absolute nicotinamide adenine dinucleotide hydrate (NADH) concentration of a liquid tissue model with the aid of a multispectral needle probe. Besides the scattering correction, this newly developed algorithm has the ability to compensate the absorbance effects of the tissue model. As an absorber new coccine (NC) is used to mimic the haemoglobin’s absorbance of human tissue, while Calcilit imitates the tissue’s scattering. The algorithm’s bases are the differences between two definite integrals of the absorbance remission and the fluorescence emission spectrum, respectively. Using this simple mathematic model, an algorithm to determine NADH concentrations between 0.5 and 2.0 mM at 2.7 wt% Calcilit and NC concentrations between 0.252 and 2.520 mM is successfully tested. This results in a mean absolute percentage error (MAPE) of 0.3% for the calculation of the Calcilit concentration and MAPEs of 3.3 and 5.0% to determine the fluorophore concentration.

The monitoring of microbiological processes using Raman spectroscopy has gained in importance over the past few years. Commercial Raman spectroscopic equipment consists of a laser, spectrometer, and fiberoptic immersion probe in direct contact with the fermentation medium. To avoid possible sterilization problems and biofilm formation on the probe tip, a large-aperture Raman probe was developed. The design of the probe enables non-contact in-line measurements through glass vessels or inspection glasses of bioreactors and chemical reactors. The practical applicability of the probe was tested during yeast fermentations by monitoring the consumption of substrate glucose and the formation of ethanol as the product. Multiple linear regression models were applied to evaluate the Raman spectra. Reference values were determined by high-performance liquid chromatography. The relative errors of prediction for glucose and ethanol were 5 and 3%, respectively. The presented Raman probe allows simple adaption to a wide range of processes in the chemical, pharmaceutical, and biotechnological industries.

In this report, a quantitative nicotinamide adenine dinucleotide hydrate (NADH) fluorescence measurement algorithm in a liquid tissue phantom using a fiber-optic needle probe is presented. To determine the absolute concentrations of NADH in this phantom, the fluorescence emission spectra at 465 nm were corrected using diffuse reflectance spectroscopy between 600 nm and 940 nm. The patented autoclavable Nitinol needle probe enables the acquisition of multispectral backscattering measurements of ultraviolet, visible, near-infrared and fluorescence spectra. As a phantom, a suspension of calcium carbonate (Calcilit) and water with physiological NADH concentrations between 0 mmol l ⁻¹ and 2.0 mmol l ⁻¹ were used to mimic human tissue. The light scattering characteristics were adjusted to match the backscattering attributes of human skin by modifying the concentration of Calcilit. To correct the scattering effects caused by the matrices of the samples, an algorithm based on the backscattered remission spectrum was employed to compensate the influence of multiscattering on the optical pathway through the dispersed phase. The monitored backscattered visible light was used to correct the fluorescence spectra and thereby to determine the true NADH concentrations at unknown Calcilit concentrations. Despite the simplicity of the presented algorithm, the root-mean-square error of prediction (RMSEP) was 0.093 mmol l ⁻¹ .

Zusammenfassung Trotz der bekannten Vorteile der Raman-Spektroskopie, wie bspw. eine höhere chemische Selektivität gegenüber Messmethoden im nahen Infrarot (NIR) oder die im Vergleich zum mittleren Infrarotbereich (MIR) niedrigen Matrixeinflüsse des Wassermoleküls, ist diese optische Messtechnik in der Online-Prozessanalysentechnik nicht weit verbreitet. Ein wesentliches Problem besteht in einem oftmals kostenintensiven Nachrüsten einer Messstelle durch den Einbau sogenannter Immersionssonden in eine produktführende Rohrleitung oder einen Behälter. Eine praktikable Alternative stellt das hier entwickelte neuartige Sondensystem dar, welches eine Strahlführung über Linsen mit relativ großen Durchmessern beinhaltet, da dieses an vorhandene Schauglasarmaturen angekoppelt werden kann. Mit diesem robusten Sondenaufbau sind Brennweiten weit über 25 mm möglich, welche Echtzeit-Messungen von außerhalb der produktführenden Leitungen durch optische Schaugläser gestatten. Die dadurch entstehenden Messoptionen werden exemplarisch am Nachweis von Ethanol durch Schaugläser unterschiedlicher Dicken sowie bei einer quantitativen Echtzeit-Verfolgung eines Propylencarbonat-Wasser-Gemisches durch eine Schauglasarmatur (Nenndruck PN 16, Nennweite DN 50) im Technikumsmaßstab untersucht. Die vorgestellte Raman-Sonde hat durch einfache Adaption an bereits vorhandene Armaturen industrieller Anlagen das Potential einer preiswerten und kontaktlosen Inline-Messlösung mit hoher Standzeit in der Prozessanalysentechnik (PAT).

A mid-infrared attenuated total reflectance (MIR-ATR) sensor has been developed for chemical reaction monitoring. The optical setup of the compact and low-priced sensor consists of an IR emitter as light source, a zinc selenide (ZnSe) ATR prism as boundary to the process, and four thermopile detectors, each equipped with an optical bandpass filter. The practical applicability was tested during esterification of ethanol and formic acid to ethyl formate and water as a model reaction with subsequent distillation. For reference analysis, a Fourier transform mid-infrared (FT-MIR) spectrometer with diamond ATR module was applied. On-line measurements using the MIR-ATR sensor and the FT-MIR spectrometer were performed in a bypass loop. The sensor was calibrated by multiple linear regression in order to link the measured absorbance in the four optical channels to the analyte concentrations. The analytical potential of the MIR-ATR sensor was demonstrated by simultaneous real-time monitoring of all four chemical substances involved in the esterification and distillation process. The temporal courses of the sensor signals are in accordance with the concentration values achieved by the commercial FT-MIR spectrometer. The standard error of prediction for ethanol, formic acid, ethyl formate, and water were 0.38 mol L ^−  1 , 0.48 mol L ^−  1 , 0.38 mol L ^−  1 , and 1.12 mol L ^−  1 , respectively. A procedure based on MIR spectra is presented to simulate the response characteristics of the sensor if the transmission ranges of the filters are varied. Using this tool analyte specific bandpass filters for a particular chemical reaction can be identified. By exchanging the optical filters, the sensor can be adapted to a wide range of processes in the chemical, pharmaceutical, and beverage industries.