Markus Brandstetter

External-Cavity Quantum Cascade Laser Spectroscopy for Mid-IR Transmission Measurements of Proteins in Aqueous Solution

In this work, we report mid-IR transmission measurements of the protein amide I band in aqueous solution at large optical paths. A tunable external-cavity quantum cascade laser (EC-QCL) operated in pulsed mode at room temperature allowed one to apply a path length of up to 38 μm, which is four times larger than that applicable with conventional FT-IR spectrometers. To minimize temperature-induced variations caused by background absorption of the ν2-vibration of water (HOH-bending) overlapping with the amide I region, a highly stable temperature control unit with relative temperature stability within 0.005 °C was developed. An advanced data processing protocol was established to overcome fluctuations in the fine structure of the emission curve that are inherent to the employed EC-QCL due to its mechanical instabilities. To allow for wavenumber accuracy, a spectral calibration method has been elaborated to reference the acquired IR spectra to the absolute positions of the water vapor absorption bands. Employing this setup, characteristic spectral features of five well-studied proteins exhibiting different secondary structures could be measured at concentrations as low as 2.5 mg mL(-1). This concentration range could previously only be accessed by IR measurements in D2O. Mathematical evaluation of the spectral overlap and comparison of second derivative spectra confirm excellent agreement of the QCL transmission measurements with protein spectra acquired by FT-IR spectroscopy. This proves the potential of the applied setup to monitor secondary structure changes of proteins in aqueous solution at extended optical path lengths, which allow experiments in flow through configuration.

Observation of particles manipulated by ultrasound in close proximity to a cone-shaped infrared spectroscopy probe

The presented investigations aimed to enhance surface sensitive infrared spectroscopy for chemical analysis by ultrasonic particle manipulation. The combination of these techniques has the potential for new measurement concepts for use in the chemical analysis of suspensions. Local increases of particle concentration brought about by ultrasound could facilitate measurements of molecular-specific infrared spectra of the suspending phase and particles independently. By changing the frequency of an ultrasonic standing wave around 2 MHz it was possible to control the position of particles in respect to the optically sensitive region of the infrared spectroscope. Results obtained with a set-up that enabled us to explore the application of an ultrasonic standing wave to push suspended particles at or into mum distances of the sensing element of an in-line fiber optic probe and subsequently retract them from there are presented. Light micrographs suggested, that the task was successfully accomplished with polystyrene beads suspended in methanol, aggregates were manipulated to and from the cut surface of the truncated, cone-shaped fibre probe tip by changes of the ultrasonic frequency between 1.85 and 1.87 MHz. Feasibility was confirmed by infrared absorption spectra recorded when PTFE particles suspended in tetrahydrofuran were used.

Remote mid-infrared photoacoustic spectroscopy with a quantum cascade laser.

We demonstrate non-contact remote photoacoustic spectroscopy in the mid-infrared region. A room-temperature-operated pulsed external-cavity quantum cascade laser is used to excite photoacoustic waves within a semitransparent sample. The ultrasonic waves are detected remotely on the opposite side of the sample using a fiber-optic Mach-Zehnder interferometer, thereby avoiding problems associated with acoustic attenuation in air. We present the theoretical background of the proposed technique and demonstrate measurements on a thin polystyrene film. The obtained absorption spectrum in the region of 1030-1230  cm(-1) is compared to a spectrum obtained by attenuated total reflection, showing reasonable agreement.

Time-resolved spectral characterization of ring cavity surface emitting and ridge-type distributed feedback quantum cascade lasers by step-scan FT-IR spectroscopy.

We present the time-resolved comparison of pulsed 2nd order ring cavity surface emitting (RCSE) quantum cascade lasers (QCLs) and pulsed 1st order ridge-type distributed feedback (DFB) QCLs using a step-scan Fourier transform infrared (FT-IR) spectrometer. Laser devices were part of QCL arrays and fabricated from the same laser material. Required grating periods were adjusted to account for the grating order. The step-scan technique provided a spectral resolution of 0.1 cm(-1) and a time resolution of 2 ns. As a result, it was possible to gain information about the tuning behavior and potential mode-hops of the investigated lasers. Different cavity-lengths were compared, including 0.9 mm and 3.2 mm long ridge-type and 0.97 mm (circumference) ring-type cavities. RCSE QCLs were found to have improved emission properties in terms of line-stability, tuning rate and maximum emission time compared to ridge-type lasers.

Ultrasound-Enhanced Attenuated Total Reflection Mid-infrared Spectroscopy In-Line Probe: Acquisition of Cell Spectra in a Bioreactor

This article presents a novel method for selective acquisition of Fourier transform infrared (FT-IR) spectra of microorganisms in-line during fermentation, using Saccharomyces cerevisiae as an example. The position of the cells relative to the sensitive region of the attenuated total reflection (ATR) FT-IR probe was controlled by combing a commercially available ATR in-line probe with contact-free, gentle particle manipulation by ultrasonic standing waves. A prototype probe was successfully constructed, assembled, and tested in-line during fed-batch fermentations of S. cerevisiae. Control over the position of the cells was achieved by tuning the ultrasound frequency: 2.41 MHz was used for acquisition of spectra of the cells (pushing frequency fp) and 1.87 MHz, for retracting the cells from the ATR element, therefore allowing spectra of the medium to be acquired. Accumulation of storage carbohydrates (trehalose and glycogen) inside the cells was induced by a lack of a nitrogen source in the feed medium. These changes in biochemical composition were visible in the spectra of the cells recorded in-line during the application of fp and could be verified by reference spectra of dried cell samples recorded off-line with a FT-IR microscope. Comparison of the cell spectra with spectra of trehalose, glycogen, glucose, and mannan, i.e., the major carbohydrates present in S. cerevisiae, and principal components analysis revealed that the changes observed in the cell spectra correlated well with the bands specific for trehalose and glycogen. This proves the applicability and capability of ultrasound-enhanced in-line ATR mid-IR spectroscopy as a real-time PAT method for the in situ monitoring of cellular biochemistry during fermentation.

Mid-infrared spectroscopic characterisation of an ultra-broadband tunable EC-QCL system intended for biomedical applications

Mid-infrared spectroscopy has been successfully applied for reagent-free clinical chemistry applications. Our aim is to design a portable bed-side system for ICU patient monitoring, based on mid-infrared absorption spectra of continuously sampled body-fluids. Robust and miniature bed-side systems can be achieved with tunable external cavity quantum cascade lasers (EC-QCL). Previously, single EC-QCL modules covering a wavenumber interval up to 250 cm-1 have been utilized. However, for broader applicability in biomedical research an extended interval around the mid-infrared fingerprint region should be accessible, which is possible with at least three or four EC-QCL modules. For such purpose, a tunable ultra-broadband system (1920 - 780 cm-1, Block Engineering) has been studied with regard to its transient emission characteristics in ns time resolution during different laser pulse widths using a VERTEX 80v FTIR spectrometer with step-scan option. Furthermore, laser emission line profiles of all four incorporated EC-QCL modules have been analysed at high spectral resolution (0.08 cm-1) and beam profiles with few deviations from the TEM 00 spatial mode have been manifested. Emission line reproducibility has been tested for various wavenumbers in step tune mode. The overall accuracy of manufacturer default wavenumber setting has been found between ± 3 cm-1 compared to the FTIR spectrometer scale. With regard to an application in clinical chemistry, theoretically achievable concentration accuracies for different blood substrates based on blood plasma and dialysate spectra previously recorded by FTIRspectrometers have been estimated taking into account the now accessible extended wavenumber interval.

Improved quantification of important beer quality parameters based on nonlinear calibration methods applied to FT-MIR spectra

AbstractDuring the production process of beer, it is of utmost importance to guarantee a high consistency of the beer quality. For instance, the bitterness is an essential quality parameter which has to be controlled within the specifications at the beginning of the production process in the unfermented beer (wort) as well as in final products such as beer and beer mix beverages. Nowadays, analytical techniques for quality control in beer production are mainly based on manual supervision, i.e., samples are taken from the process and analyzed in the laboratory. This typically requires significant lab technicians efforts for only a small fraction of samples to be analyzed, which leads to significant costs for beer breweries and companies. Fourier transform mid-infrared (FT-MIR) spectroscopy was used in combination with nonlinear multivariate calibration techniques to overcome (i) the time consuming off-line analyses in beer production and (ii) already known limitations of standard linear chemometric methods, like partial least squares (PLS), for important quality parameters Speers et al. (J I Brewing. 2003;109(3):229–235), Zhang et al. (J I Brewing. 2012;118(4):361–367) such as bitterness, citric acid, total acids, free amino nitrogen, final attenuation, or foam stability. The calibration models are established with enhanced nonlinear techniques based (i) on a new piece-wise linear version of PLS by employing fuzzy rules for local partitioning the latent variable space and (ii) on extensions of support vector regression variants (𝜖-PLSSVR and ν-PLSSVR), for overcoming high computation times in high-dimensional problems and time-intensive and inappropriate settings of the kernel parameters. Furthermore, we introduce a new model selection scheme based on bagged ensembles in order to improve robustness and thus predictive quality of the final models. The approaches are tested on real-world calibration data sets for wort and beer mix beverages, and successfully compared to linear methods, showing a clear out-performance in most cases and being able to meet the model quality requirements defined by the experts at the beer company. FigureWorkflow for calibration of non-Linear model ensembles from FT-MIR spectra in beer production 

Ultrasonic Manipulation of Yeast Cells in Suspension for Absorption Spectroscopy with an Immersible Mid-Infrared Fiberoptic Probe

Recent advances in combining ultrasonic particle manipulation with attenuated total reflection infrared spectroscopy of yeast suspensions are presented. Infrared spectroscopy provides highly specific molecular information about the sample. It has not been applicable to in-line monitoring of cells during fermentation, however, because positioning cells in the micron-thin measurement region of the attenuated total reflection probe was not possible. Ultrasonic radiation forces exerted on suspended particles by an ultrasonic standing wave can result in the buildup of agglomerates in the nodal planes, hence enabling the manipulation of suspended cells on the microscopic scale. When a chamber setup and a prototype in-line applicable probe were used, successful control over the position of the yeast cells relative to the attenuated total reflection sensor surface could be proven. Both rate of increase and maximum mid-infrared absorption of yeast-specific bands during application of a pushing frequency (chamber setup: 1.863 MHz, in-line probe: 1.990 MHz) were found to correlate with yeast cell concentration.

Measures for optimizing pulsed EC-QC laser spectroscopy of liquids and application to multi-analyte blood analysis

We employed a broadly tunable pulsed external cavity (EC)-QC laser with a spectral tuning range from 1030 cm-1 to 1230 cm-1 and a tuning speed of 166 cm-1/s for direct absorption spectroscopy of aqueous solutions. The laser offered spectral power densities of up to four orders of magnitude higher than available with a conventional FTIR spectrometer. Therefore, a portable demonstration system with a large optical path length transmission flow cell (165 μm) could be realized preventing clogging of the flow cell. In pulsed mode an EC-QC laser provides significantly higher peak power levels than in continuous-wave mode, but pulse-to-pulse intensity variations, intra-pulse mode hops and mechanical imperfections of the scanning mechanism significantly impair the quality of resulting absorbance spectra. This article reports on measures which we found appropriate to reduce the initially high noise level of EC-QC laser absorbance spectra. These measures include a spectral self-referencing algorithm that makes use of the inherent structure of the EC-QC lasers gain curve to correct laser instabilities, as well as Fourier filtering, among others. This enabled us to derive infrared spectra which were finally useful for quantitative analysis in blood plasma samples. Finally, with the appropriate measures in place and using partial least squares regression analysis it was possible to simultaneously quantify 6 blood analytes from a single physical measurement of a 200 μL blood sample. This proves the potential of EC-QC lasers for practical application in clinical point of care analysis.

Mid-Infrared Standoff Spectroscopy Using a Supercontinuum Laser with Compact Fabry–Pérot Filter Spectrometers:

Mid-infrared (MIR) supercontinuum (SC) lasers are an attractive new option in the field of IR spectroscopy, especially for standoff detection. Supercontinuum radiation unites high brightness, high spatial coherence, and broadband spectral coverage, thereby surpassing thermal IR sources and challenging quantum cascade lasers. The employed SC source operates in the spectral region of 1.2–4.6 µm, filling the spectral gap where quantum cascade lasers lack broader availability. In this work, the SC radiation was recorded by compact Fabry–Pérot filter spectrometers ideally suited for sensitive standoff detection with real-time capability. The noise performance of the setup and measurements of different substances at standoff distances are presented, e.g., of different paints on a metal surface and an explosive precursor. Furthermore, the real-time capability of the setup is demonstrated by monitoring the evaporation of liquid 2-propanol.