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.

Optical systems applied for tissue analysis are primarily based on single spectroscopic techniques. This paper however presents a multispectral backscattering sensor designed for in vivo application by a specially formed probe tip which allows side by side monitoring of ultraviolet, visible, near-infrared and fluorescence spectra. The practical applicability of the measurement system was demonstrated in vitro (muscle and adipose tissue) and in vivo in an animal model (mouse). By comparing associated measuring changes in biochemical, physical-morphological and colorimetric values this procedure allows a differentiation between healthy, marginal and malignant tissue.

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 2.0mM at 2.7 wt% Calcilit and NC concentrations between 0.252 and 2.520mM 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.

A mid-infrared (MIR) sensor using the attenuated total reflection (ATR) technique has been developed for real-time monitoring in biotechnology. The MIR-ATR sensor consists of an IR emitter as light source, a zinc selenide ATR prism as boundary to the process, and four thermopile detectors, each equipped with an optical bandpass filter. The suitability of the sensor for practical application was tested during aerobic batch-fermentations of Saccharomyces cerevisiae by simultaneous monitoring of glucose and ethanol. The performance of the sensor was compared to a commercial Fourier transform mid-infrared (FT-MIR) spectrometer by on-line measurements in a bypass loop. Sensor and spectrometer were calibrated by multiple linear regression (MLR) in order to link the measured absorbance in the transmission ranges of the four optical sensor channels to the analyte concentrations. For reference analysis, high-performance liquid chromatography (HPLC) was applied. Process monitoring using the sensor yielded in standard errors of prediction (SEP) of 6.15 g/L and 1.36 g/L for glucose and ethanol. In the case of the FT-MIR spectrometer the corresponding SEP values were 4.34 and 0.61 g/L, respectively. The advantages of optical multi-channel mid-infrared sensors in comparison to FT-MIR spectrometer setups are the compactness, easy process implementation and lower price. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

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.

A multi-channel fluorescence sensor has been developed for process monitoring and fluorescence diagnostics. It comprises a fiber-optic set-up with an immersion probe and an intensity-modulated high power ultraviolet light-emitting diode as a light source for fluorescence excitation. By applying an electronic lock-in procedure, fluorescence signals are selectively detectable at ambient light levels of 1000 000 times higher intensity. The sensor was designed to be compact, low cost and easily adaptable to a wide field of application. The set-up was used to simultaneously monitor three important metabolic fluorophores: NAD(P)H, flavins and porphyrins during the cultivation of a baker's yeast. Moreover, the accumulation and degradation kinetics of protoporphyrin IX induced by 5-aminolevulinic acid on the skin could be recorded by the sensor. The detection limit for protoporphyrin IX was determined to be 4 x 10(-11) mol L-1. The linear signal amplification of the sensor and time courses of fluorescence signals monitored during yeast fermentations were validated using a commercial CCD spectrometer. The robust and flexible set-up of the fiber-optic measurement system promises easy implementation of this non-invasive analytical tool to fluorescence monitoring and diagnostics in R&D and production.

Monitoring of microbiological processes using optical sensors and spectrometers has gained in importance over the past few years due to its advantage in enabling non-invasive on-line analysis. Near-infrared (NIR) and mid-infrared (MIR) spectrometer set-ups in combination with multivariate calibrations have already been successfully employed for the simultaneous determination of different metabolites in microbiological processes. Photometric sensors, in addition to their low price compared to spectrometer set-ups, have the advantage of being compact and are easy to calibrate and operate. In this work, the detection of ethanol and CO2 in the exhaust gas during aerobic yeast fermentation was performed by two photometric gas analyzers, and dry yeast biomass was monitored using a fiber optic backscatter set-up. The optical sensors could be easily fitted to the bioreactor and exhibited high robustness during measuring. The ethanol content of the fermentation broth was monitored on-line by measuring the ethanol concentration in the fermentation exhaust and applying a conversion factor. The vapor/liquid equilibrium and the associated conversion factor strongly depend on the process parameter temperature but not on aeration and stirring rate. Dry yeast biomass was determined in-line by a backscattering signal applying a linear calibration. An on-line balance with a recovery rate of 95–97% for carbon was achieved with the use of three optical sensors (two infrared gas analyzers and one fiber optic backscatter set-up).

L'invention concerne un dispositif permettant de reguler des processus de synthese chimique a l'aide d'une determination en ligne de la concentration d'au moins une substance d'un melange reactionnel, ladite substance influant et/ou donnant une indication sur le deroulement d'un processus de synthese. Ledit dispositif comporte au moins les composants suivants : (a) au moins une installation destinee a prelever au moins un echantillon du melange reactionnel ; (b) au moins une installation concue pour loger et le cas echeant pour preparer le ou les echantillons ; (c) au moins une installation permettant de determiner, dans le ou les echantillons, un parametre lie a la concentration d'une substance influant et/ou donnant une indication sur le deroulement du processus de synthese ; (d) au moins une installation concue pour interpreter les resultats de la determination du ou des parametres selon (c), et le cas echeant, pour les memoriser et les transformer en signaux destines a reguler un dispositif de synthese ; (e) au moins une installation concue pour coordonner le deroulement d'une determination de concentration en ligne faisant appel au moins aux installations (a) a (d).

The invention relates to a device for controlling chemical synthesis processes by determining online the concentration of at least one substance in a reaction mixture, which influences and/or indicates the progression of a synthesis process, wherein the device comprises the following components: (a) at least one device suitable for taking at least one sample from the reaction mixture; (b) at least one device suitable for receiving and optionally processing the at least one sample; (c) at least one device suitable for determining a proportional parameter relative to the concentration of at least one substance which influences and/or indicates the progression of the synthesis process in the at least one sample; (d) at least one device suitable for evaluating the results of the determination of the at least one parameter according to (c) and optionally storing and converting said results into signals suitable for controlling a synthesis device; (e) at least one device suitable for coordinating the progression of online concentration determination by at least the devices (a) to (d).