Infrared nanoscopy and tomography of intracellular structures.

Although techniques such as fluorescence-based super-resolution imaging or confocal microscopy simultaneously gather both morphological and chemical data, these techniques often rely on the use of localized and chemically specific markers. To eliminate this flaw, we have developed a method of examining cellular cross sections using the imaging power of scattering-type scanning near-field optical microscopy and Fourier-transform infrared spectroscopy at a spatial resolution far beyond the diffraction limit. Herewith, nanoscale surface and volumetric chemical imaging is performed using the intrinsic contrast generated by the characteristic absorption of mid-infrared radiation by the covalent bonds. We employ infrared nanoscopy to study the subcellular structures of eukaryotic (Chlamydomonas reinhardtii) and prokaryotic (Escherichia coli) species, revealing chemically distinct regions within each cell such as the microtubular structure of the flagellum. Serial 100 nm-thick cellular cross-sections were compiled into a tomogram yielding a three-dimensional infrared image of subcellular structure distribution at 20 nm resolution. The presented methodology is able to image biological samples complementing current fluorescence nanoscopy but at less interference due to the low energy of infrared radiation and the absence of labeling.

Rapid brain structure and tumour margin detection on whole frozen tissue sections by fast multiphotometric mid-infrared scanning.

Frozen section analysis is a frequently used method for examination of tissue samples, especially for tumour detection. In the majority of cases, the aim is to identify characteristic tissue morphologies or tumour margins. Depending on the type of tissue, a high number of misdiagnoses are associated with this process. In this work, a fast spectroscopic measurement device and workflow was developed that significantly improves the speed of whole frozen tissue section analyses and provides sufficient information to visualize tissue structures and tumour margins, dependent on their lipid and protein molecular vibrations. That optical and non-destructive method is based on selected wavenumbers in the mid-infrared (MIR) range. We present a measuring system that substantially outperforms a commercially available Fourier Transform Infrared (FT-IR) Imaging system, since it enables acquisition of reduced spectral information at a scan field of 1 cm2 in 3 s, with a spatial resolution of 20 µm. This allows fast visualization of segmented structure areas with little computational effort. For the first time, this multiphotometric MIR system is applied to biomedical tissue sections. We are referencing our novel MIR scanner on cryopreserved murine sagittal and coronal brain sections, especially focusing on the hippocampus, and show its usability for rapid identification of primary hepatocellular carcinoma (HCC) in mouse liver.

Dual Color Imaging from a Single BF2-Azadipyrromethene Fluorophore Demonstrated in vivo for Lymph Node Identification.

Dual emissions at ~700 and 800 nm have been achieved from a single NIR-AZA fluorophore 1 by establishing parameters in which it can exist in either its isolated molecular or aggregated states. Dual near infrared (NIR) fluorescence color lymph node (LN) mapping with 1 was achieved in a large-animal porcine model, with injection site, channels and nodes all detectable at both 700 and 800 nm using a preclinical open camera system. The fluorophore was also compatible with imaging using two clinical instruments for fluorescence guided surgery. Methods: An NIR-AZA fluorophore with hydrophilic and phobic features was synthesised in a straightforward manner and its aggregation properties characterised spectroscopically and by TEM imaging. Toxicity was assessed in a rodent model and dual color fluorescence imaging evaluated by lymph node mapping in a large animal porcine models and in ex-vivo human tissue specimen. Results: Dual color fluorescence imaging has been achieved in the highly complex biomedical scenario of lymph node mapping. Emissions at 700 and 800 nm can be achieved from a single fluorophore by establishing molecular and aggregate forms. Fluorophore was compatible with clinical systems for fluorescence guided surgery and no toxicity was observed in high dosage testing. Conclusion: A new, biomedical compatible form of NIR-dual emission wavelength imaging has been established using a readily accessible fluorophore with significant scope for clinical translation.

Micro Freeze-Dryer and Infrared-Based PAT: Novel Tools for Primary Drying Design Space Determination of Freeze-Drying Processes.

PURPOSE: Present (i) an infrared (IR)-based Process Analytical Technology (PAT) installed in a lab-scale freeze-dryer and (ii) a micro freeze-dryer (MicroFD®) as effective tools for freeze-drying design space calculation of the primary drying stage. METHODS: The case studies investigated are the freeze-drying of a crystalline (5% mannitol) and of an amorphous (5% sucrose) solution processed in 6R vials. The heat (Kv) and the mass (Rp) transfer coefficients were estimated: tests at 8, 13 and 26 Pa were carried out to assess the chamber pressure effect on Kv. The design space of the primary drying stage was calculated using these parameters and a well-established model-based approach. The results obtained using the proposed tools were compared to the ones in case Kv and Rp were estimated in a lab-scale unit through gravimetric tests and a thermocouple-based method, respectively. RESULTS: The IR-based method allows a non-gravimetric estimation of the Kv values while with the micro freeze-dryer gravimetric tests require a very small number of vials. In both cases, the obtained values of Kv and Rp, as well as the resulting design spaces, were all in very good agreement with those obtained in a lab-scale unit through the gravimetric tests (Kv) and the thermocouple-based method (Rp). CONCLUSIONS: The proposed tools can be effectively used for design space calculation in substitution of other well-spread methods. Their advantages are mainly the less laborious Kv estimation process and, as far as the MicroFD® is concerned, the possibility of saving time and formulation material when evaluating Rp.

Implementing an integrated multi-technology platform for drug checking: Social, scientific, and technological considerations.

The illicit drug overdose crisis in North America continues to devastate communities with fentanyl detected in the majority of illicit drug overdose deaths. The COVID-19 pandemic has heightened concerns of even greater unpredictability in the drug supplies and unprecedented rates of overdoses. Portable drug-checking technologies are increasingly being integrated within overdose prevention strategies. These emerging responses are raising new questions about which technologies to pursue and what service models can respond to the current risks and contexts. In what has been referred to as the epicenter of the overdose crisis in Canada, a multi-technology platform for drug checking is being piloted in community settings using a suite of chemical analytical methods to provide real-time harm reduction. These include infrared absorption, Raman scattering, gas chromatography with mass spectrometry, and antibody-based test strips. In this Perspective, we illustrate some advantages and challenges of using multiple techniques for the analysis of the same sample, and provide an example of a data analysis and visualization platform that can unify the presentation of the results and enable deeper analysis of the results. We also highlight the implementation of a various service models that co-exist in a research setting, with particular emphasis on the way that drug checking technicians and harm reduction workers interact with service users. Finally, we provide a description of the challenges associated with data interpretation and the communication of results to a diverse audience.

Portable NIR spectrometer for quick identification of fat bloom in chocolates.

Cocoa butter provides desirable sensory properties to chocolates; however, the exposure of chocolate to temperature variations during transportation and/or storage can lead to changes in the polymorphic form of butter, with the appearance of a dull-white film on the chocolate surface, known as fat bloom. This study investigated the use of a portable NIR spectrometer combined with chemometric tools to discriminate milk chocolate, white chocolate, 40% cocoa chocolate, and 70% cocoa chocolate samples, which were subjected to temperature abuse for 6 hours. The PCA allowed separating the samples into three classes: control at 20 °C, chocolate subjected to 35 °C, and chocolate subjected to 40 °C, for each type of chocolate studied. The PLS-DA models provided sensibility, specificity, and accuracy values in the range of 80 to 100%, and allowed identifying the wavelengths associated with the different chocolates that most impacted the construction of the models.

Characterization and identification of ascorbyl methylsilanol pectinate for cosmetic formulations application

The ascorbyl methylsilanol pectinate (AMP) presents the same functional properties of ascorbic acid (AA). Besides antioxidant and depigmentant activity, the AMP presents silanol in its chemical structure. The aim of this work was to characterize and indentify the AMP alone and in cosmetic formulations. The following techniques were employed: Fourier Transform Infrared Spectrophotometry, particle size distributions, in vitro antioxidant activity with 2.2-diphenyl-1-picrylhydrazyl (DPPH) and Oxigen Radical Absorbance Capacity Assay and High Performace Liquid Chromatography (HPLC) (developed and validated method) for the active ingredient; Microscopy, HPLC and Normal Stability Assay (NSA) for the emulsions. Particle size distributions results showed that the average size of AMP was 1.0 µm and polydispersity index was 0.1. In DPPH assay AA and AMP were statistically the same. The value of ORAC obtained for AMP was 0.74 and for AA in the literature was 0.95. In the NSA the formulations were stable in conditions of 5.0 and 45.0 ± 2.0 ºC for 90 days. Adequate stability at ambient temperature out of reach of light was also observed. Thus, this works presented an acceptable method for quantification of AMP alone and in cosmetic formulations. AMP was an adequate choice for the incorporation in emulsions with antioxidant efficacy.

Multimodal Imaging Mass Spectrometry: Next Generation Molecular Mapping in Biology and Medicine.

Imaging mass spectrometry has become a mature molecular mapping technology that is used for molecular discovery in many medical and biological systems. While powerful by itself, imaging mass spectrometry can be complemented by the addition of other orthogonal, chemically informative imaging technologies to maximize the information gained from a single experiment and enable deeper understanding of biological processes. Within this review, we describe MALDI, SIMS, and DESI imaging mass spectrometric technologies and how these have been integrated with other analytical modalities such as microscopy, transcriptomics, spectroscopy, and electrochemistry in a field termed multimodal imaging. We explore the future of this field and discuss forthcoming developments that will bring new insights to help unravel the molecular complexities of biological systems, from single cells to functional tissue structures and organs.

Metasurface with metallic nanoantennas and graphene nanoslits for sensing of protein monolayers and sub-monolayers.

Biomolecule sensing plays an important role in both fundamental biological studies and medical diagnostic applications. Infrared (IR) spectroscopy presents opportunities for sensing biomolecules as it allows their fingerprints to be determined by directly measuring their absorption spectra. However, the detection of biomolecules at low concentrations is difficult with conventional IR spectroscopy due to signal-to-noise considerations. This has led to recent interest on the use of nanostructured surfaces to boost the signals from biomolecules in a method termed surface enhanced infrared spectroscopy. So far, efforts have largely involved the use of metallic nanoantennas (which produce large field enhancement) or graphene nanostructures (which produce strong field confinement and provide electrical tunability). Here, we propose a nanostructured surface that combines the large field enhancement of metallic nanoantennas with the strong field confinement and electrical tunability of graphene plasmons. Our device consists of an array of plasmonic nanoantennas and graphene nanoslits on a resonant substrate. We perform systematic electromagnetic simulations to quantify the sensing performance of the proposed device and show that it outperforms designs in which only plasmons from metallic nanoantennas or plasmons from graphene are utilized. These investigations consider the model system of a representative protein-goat anti-mouse immunoglobulin G (IgG) - in monolayer or sub-monolayer form. Our findings provide guidance for future biosensors for the sensitive quantification and identification of biomolecules.

Perspective on Thin Film Waveguides for on-Chip Mid-Infrared Spectroscopy of Liquid Biochemical Analytes.

Miniaturized spectrometers offering low cost, low reagent consumption, high throughput, sensitivity and automation are the future of sensing and have significant applications in environmental monitoring, food safety, biotechnology, pharmaceuticals, and healthcare. Midinfrared (MIR) spectroscopy employing complementary metal oxide semiconductor (CMOS) compatible thin film waveguides and microfluidics shows great promise toward highly integrated and robust detection tools and liquid handling. This perspective provides an overview of the emergence of thin film optical waveguides used for evanescent field sensing of liquid chemical and biological samples for MIR absorption spectroscopy. The state of the art of new material and waveguide systems used for spectroscopic measurements in the MIR is presented. An outlook on the advantages and future of waveguide-based MIR spectroscopy for application in clinical settings for point-of-care biochemical analysis is discussed.

Advances in Mid-Infrared Spectroscopy-Based Sensing Techniques for Exhaled Breath Diagnostics.

Human exhaled breath consists of more than 3000 volatile organic compounds, many of which are relevant biomarkers for various diseases. Although gas chromatography has been the gold standard for volatile organic compound (VOC) detection in exhaled breath, recent developments in mid-infrared (MIR) laser spectroscopy have led to the promise of compact point-of-care (POC) optical instruments enabling even single breath diagnostics. In this review, we discuss the evolution of MIR sensing technologies with a special focus on photoacoustic spectroscopy, and its application in exhaled breath biomarker detection. While mid-infrared point-of-care instrumentation promises high sensitivity and inherent molecular selectivity, the lack of standardization of the various techniques has to be overcome for translating these techniques into more widespread real-time clinical use.

InGaAs Membrane Waveguide: A Promising Platform for Monolithic Integrated Mid-Infrared Optical Gas Sensor.

Mid-infrared (mid-IR) absorption spectroscopy based on integrated photonic circuits has shown great promise in trace-gas sensing applications in which the mid-IR radiation directly interacts with the targeted analyte. In this paper, considering monolithic integrated circuits with quantum cascade lasers (QCLs) and quantum cascade detectors (QCDs), the InGaAs-InP platform is chosen to fabricate passive waveguide gas sensing devices. Fully suspended InGaAs waveguide devices with holey photonic crystal waveguides (HPCWs) and subwavelength grating cladding waveguides (SWWs) are designed and fabricated for mid-infrared sensing at λ = 6.15 µm in the low-index contrast InGaAs-InP platform. We experimentally detect 5 ppm ammonia with a 1 mm long suspended HPCW and separately with a 3 mm long suspended SWW, with propagation losses of 39.1 and 4.1 dB/cm, respectively. Furthermore, based on the Beer-Lambert infrared absorption law and the experimental results of discrete components, we estimated the minimum detectable gas concentration of 84 ppb from a QCL/QCD integrated SWW sensor. To the best of our knowledge, this is the first demonstration of suspended InGaAs membrane waveguides in the InGaAs-InP platform at such a long wavelength with gas sensing results. Also, this result emphasizes the advantage of SWWs to reduce the total transmission loss and the size of the fully integrated device's footprint by virtue of its low propagation loss and TM mode compatibility in comparison to HPCWs. This study enables the possibility of monolithic integration of quantum cascade devices with TM polarized characteristics and passive waveguide sensing devices for on-chip mid-IR absorption spectroscopy.

Nanoscale investigation of human skin and study of skin penetration of Janus nanoparticles.

Janus nanoparticles (JNP) are innovative nanocarriers with an interesting pharmaceutical and cosmetic potential. They are characterized by the presence of a lipid compartment associated with an aqueous compartment delimited by a phospholipid bilayer containing phospholipids and non-ionic surfactants. The hydrodynamic diameter of JNP varies between 150 and 300 nm. The purpose of this study was to answer the following questions: after cutaneous application, are JNP penetrating? If so, how deep? And in which state, intact or degraded? It was essential to understand these phenomena in order to control the rate and kinetics of diffusion of active ingredients, which can be encapsulated in this vehicle for pharmaceutical or cosmetic purposes. An innovative technique called AFM-IR, was used to elucidate the behavior of JNP after cutaneous application. This instrument, coupling atomic force microscopy and IR spectroscopy, allowing to perform chemical analysis at the nanometer scale thanks to local absorption measurements. The identification of organic molecules at the nanoscale is possible without any labelling. Before cutaneous application of JNP, the nano-structure of untreated human skin was investigated with AFM-IR. Then, in vitro human skin penetration of JNP was studied using Franz cells, and AFM-IR allowed us to perform ultra-local information investigations.

Two-dimensional infrared spectroscopy: an emerging analytical tool?

Ultrafast two-dimensional infrared (2D-IR) spectroscopy has provided valuable insights into biomolecular structure and dynamics, but recent progress in laser technology and data analysis methods have demonstrated the potential for high throughput 2D-IR measurements and analytical applications. Using 2D-IR as an analytical tool requires a different approach to data collection and analysis compared to pure research applications however and, in this review, we highlight progress towards usage of 2D-IR spectroscopy in areas relevant to biomedical, pharmaceutical and analytical molecular science. We summarise the technical and methodological advances made to date and discuss the challenges that still face 2D-IR spectroscopy as it attempts to transition from the state-of-the-art laser laboratory to the standard suite of analytical tools.

Milk analysis using milk analyzers in a standardized setting (MAMAS) study: A multicentre quality initiative.

BACKGROUND: Human milk analyzers are increasingly used to rapidly measure the macronutrient content in breast milk for individual target fortification, to reduce the risk of postnatal growth restriction. However, many milk analyzers are used without calibration, validation or quality assurance. AIMS: To investigate measurement quality between different human milk analyzers, to test whether accuracy and precision of devices can be improved by establishing individual calibration curves, and to assess long-term stability of measurements, following good clinical laboratory practice (GCLP). METHODS: Sets of identical breast milk samples were sent to 13 participating centres in North America and Europe, for a total of 15 devices. The study included 3 sets of samples: A) initial assessment of the device's performance consisting of 10 calibration samples with random replicates; B) long term stability and quality control consisting of 2 batches of samples to be measured every time before the device is used, over 6 months; C) ring trial consisting of 2 samples to be measured monthly. The devices tested were Unity SpectraStar (n = 5) and MIRIS Human Milk Analyzer (n = 10). RESULTS: There are significant variations in accuracy and precision between different milk analyzers' fat, protein and lactose measurements. However, the accuracy of measurements can be improved by establishing individual correction algorithms. Repeated measurements are more robust when coming from a larger batch volume. Long term stability also varies between devices. CONCLUSION: The variations in measurements between devices are clinically significant and would impact both daily dietary prescriptions, and the outcomes of clinical studies assessing the effect of targeted adjustment of nutrient intake in preterm babies. This study shows that it is crucial to follow GCLP when using milk analyzers to ensure proper measurement of macronutrients, similar to what is required of other medical devices.

IR action spectroscopy of glycosaminoglycan oligosaccharides.

Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm-1 for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy. Graphical abstract.

Féry Infrared Spectrometer for Single-Shot Analysis of Protein Dynamics.

Current submillisecond time-resolved broad-band infrared spectroscopy, one of the most frequently used techniques for studying structure-function relationships in life sciences, is typically limited to fast-cycling reactions that can be repeated thousands of times with high frequency. Notably, a majority of chemical and biological processes do not comply with this requirement. For example, the activation of vertebrate rhodopsin, a prototype of many protein receptors in biological organisms that mediate basic functions of life, including vision, smell, and taste, is irreversible. Here we present a dispersive single-shot Féry spectrometer setup that extends such spectroscopy to irreversible and slow-cycling systems by exploiting the unique properties of brilliant synchrotron infrared light combined with an advanced focal plane detector array embedded in a dispersive optical concept. We demonstrate our single-shot method on microbial actinorhodopsin with a slow photocycle and on vertebrate rhodopsin with irreversible activation.

Investigations into the use of handheld near-infrared spectrometer and novel semi-automated data analysis for the determination of protein content in different cultivars of Panicum miliaceumL.

A set of 42 millet (panicum miliaceum L.) samples was investigated for its protein content using standard Kjeldahl analysis and near-infrared spectroscopy. The performance of three handheld spectrometers was compared to a benchtop instrument. The used spectrometers operate in different regions of the NIR, which gives interesting insights into the applicability of each region. Additionally, semi-automated, consumer-oriented multivariate data analysis was compared to sophisticated data evaluation. The performance of the near-infrared instruments was compared using important statistical parameters of the established cross- and test set validated partial least squares regression (PLS-R) models. Milled and intact samples were analysed, in order to further evaluate the importance of homogeneity. The results showed that the benchtop spectrometer is capable of accurately analysing protein content of millet grains, with root mean square error (RMSEP) values for milled and intact grains of approximately 0.5%. Two PLS-R models of handheld instruments also yielded good results for milled grains with RMSEP values of about 0.6%. The semi-automated multivariate data analysis showed some drawbacks compared to standard data processing software. For intact grains, however, similar results could be achieved.

Far-Field Super-Resolution Vibrational Spectroscopy.

Potential label-free alternatives to super-resolution fluorescence techniques have been the focus of considerable research due to the challenges intrinsic in the reliance on fluorescent tags. In this Feature, we discuss efforts to develop super-resolution techniques based on vibrational spectroscopies and address possible sample applications as well as future potential resolution enhancements.

An Acoustic Trap for Bead Injection Attenuated Total Reflection Infrared Spectroscopy.

In this work, we introduce a system combining an acoustic trap for bead injection with attenuated total reflection (ATR) infrared (IR) spectroscopy. By mounting an acoustofluidic cell hosting an ultrasound source on top of a custom-built ATR fixture we were able to trap beads labeled with the enzyme alkaline phosphatase without requiring any mechanical retention elements. Sequential injection analysis was employed for reproducible sample handling and bead injection into the acoustic trap. To showcase potential applications of the presented setup for kinetic studies, we monitored the conversion of p-nitrophenylphosphate into p-nitrophenol and phosphate via beads carrying the immobilized enzyme using ATR-IR spectroscopy. Retaining the labeled beads via ultrasound particle manipulation resulted in excellent experimental reproducibility (relative standard deviation, 3.91%). It was demonstrated that trapped beads remained stably restrained with up to eight cell volumes of liquid passing through the acoustofluidic cell. Beads could be discarded in a straightforward manner by switching off the ultrasound, in contrast to systems containing mechanical retention elements, which require backflushing. Multiple experiments were performed by employing different substrate concentrations with the same batch of trapped beads as well as varying the amount of enzyme present in the cell, enabling enzyme kinetic studies and emphasizing the application of the proposed setup in studies where enzymatic reuse is desired. This proves the potential of the acoustic trap combined with ATR-IR spectroscopy to monitor the activity of immobilized enzymes and its ability to perform complex bead-based assays.