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RAMANMETRIX: a delightful way to analyze Raman spectra

ArXiv:2201.07586 [Physics, Stat]
Darina Storozhuk, Oleg Ryabchykov, Juergen Popp, Thomas Bocklitz

Although Raman spectroscopy is widely used for the investigation of biomedical samples and has a high potential for use in clinical applications, it is not common in clinical routines. One of the factors that obstruct the integration of Raman spectroscopic tools into clinical routines is the complexity of the data processing workflow. Software tools that simplify spectroscopic data handling may facilitate such integration by familiarizing clinical experts with the advantages of Raman spectroscopy. Here, RAMANMETRIX is introduced as a user-friendly software with an intuitive web-based graphical user interface (GUI) that incorporates a complete workflow for chemometric analysis of Raman spectra, from raw data pretreatment to a robust validation of machine learning models. The software can be used both for model training and for the application of the pretrained models onto new data sets. Users have full control of the parameters during model training, but the testing data flow is frozen and does not require additional user input. RAMANMETRIX is available in two versions: as standalone software and web application. Due to the modern software architecture, the computational backend part can be executed separately from the GUI and accessed through an application programming interface (API) for applying a preconstructed model to the measured data. This opens up possibilities for using the software as a data processing backend for the measurement devices in real-time. The models preconstructed by more experienced users can be exported and reused for easy one-click data preprocessing and prediction, which requires minimal interaction between the user and the software. The results of such prediction and graphical outputs of the different data processing steps can be exported and saved.

Supporting files

Dataset used as an example: Sample-Size Planning for Multivariate Data: A Raman-Spectroscopy-Based Exampleopen in new window, N. Ali, S. Girnus, P. Rösch, J. Popp, T. Bocklitz, , Anal. Chem. 90 (2018) 12485–12492.

Metadata fileopen in new window

Training reportopen in new window


User publications

Use of Polymers as Wavenumber Calibration Standards in Deep-UVRR

Pistiki, Aikaterini, Oleg Ryabchykov, Thomas W. Bocklitz, Petra Rösch, and Jürgen Popp.
In: Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 287 (15 February 2023): 122062

Deep-UV resonance Raman spectroscopy (UVRR) allows the classification of bacterial species with high accuracy and is a promising tool to be developed for clinical application. For this attempt, the optimization of the wavenumber calibration is required to correct the overtime changes of the Raman setup. In the present study, different polymers were investigated as potential calibration agents. The ones with many sharp bands within the spectral range 400–1900 cm−1 were selected and used for wavenumber calibration of bacterial spectra. Classification models were built using a training cross-validation dataset that was then evaluated with an independent test dataset obtained after 4 months. Without calibration, the training cross-validation dataset provided an accuracy for differentiation above 99 % that dropped to 51.2 % after test evaluation. Applying the test evaluation with PET and Teflon calibration allowed correct assignment of all spectra of Gram-positive isolates. Calibration with PS and PEI leads to misclassifications that could be overcome with majority voting. Concerning the very closely related and similar in genome and cell biochemistry Enterobacteriaceae species, all spectra of the training cross-validation dataset were correctly classified but were misclassified in test evaluation. These results show the importance of selecting the most suitable calibration agent in the classification of bacterial species and help in the optimization of the deep-UVRR technique.

Specific Intracellular Signature of SARS-CoV-2 Infection Using Confocal Raman Microscopy’

Salehi, Hamideh, Anuradha Ramoji, Said Mougari, Peggy Merida, Aymeric Neyret, Jurgen Popp, Branka Horvat, Delphine Muriaux, and Frederic Cuisinier.
In: Communications Chemistry 5, no. 1 (25 July 2022): 1–10.

SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.

Comparison of Different Label-Free Raman Spectroscopy Approaches for the Discrimination of Clinical MRSA and MSSA Isolates

Aikaterini Pistiki, Stefan Monecke, Haodong Shen, Oleg Ryabchykov, Thomas W. Bocklitz, Petra Rösch, Ralf Ehricht, Jürgen Popp
In: Microbiology Spectrum (22 August 2022)

Methicillin-resistant Staphylococcus aureus (MRSA) is classified as one of the priority pathogens that threaten human health. Resistance detection with conventional microbiological methods takes several days, forcing physicians to administer empirical antimicrobial treatment that is not always appropriate. A need exists for a rapid, accurate, and cost-effective method that allows targeted antimicrobial therapy in limited time. In this pilot study, we investigate the efficacy of three different label-free Raman spectroscopic approaches to differentiate methicillin-resistant and -susceptible clinical isolates of S. aureus (MSSA). Single-cell analysis using 532 nm excitation was shown to be the most suitable approach since it captures information on the overall biochemical composition of the bacteria, predicting 87.5% of the strains correctly. UV resonance Raman microspectroscopy provided a balanced accuracy of 62.5% and was not sensitive enough in discriminating MRSA from MSSA. Excitation of 785 nm directly on the petri dish provided a balanced accuracy of 87.5%. However, the difference between the strains was derived from the dominant staphyloxanthin bands in the MRSA, a cell component not associated with the presence of methicillin resistance. This is the first step toward the development of label-free Raman spectroscopy for the discrimination of MRSA and MSSA using single-cell analysis with 532 nm excitation.

Label-free differentiation of clinical E. coli and Klebsiella isolates with Raman spectroscopy

Nakar, Amir, Aikaterini Pistiki, Oleg Ryabchykov, Thomas Bocklitz, Petra Rösch, and Jürgen Popp.
In: Journal of Biophotonics (7 April 2022)

Raman spectroscopy is a promising spectroscopic technique for microbiological diagnostics. In routine diagnostic, the differentiation of pathogens of the Enterobacteriaceae family remain challenging. In this study, Raman spectroscopy was applied for the differentiation of 24 clinical E. coli, Klebsiella pneumoniae and Klebsiella oxytoca isolates. Spectra were collected with two spectroscopic approaches: UV-Resonance Raman spectroscopy (UVRR) and single-cell Raman microspectroscopy with 532 nm excitation. A description of the different biochemical profiles provided by the different excitation wavelengths was performed followed by machine-learning models for the classification at the genus and species levels. UVRR was shown to outperform 532 nm excitation, enabling correct classification at the genus level of 23/24 isolates. Furthermore, for the first time, Klebsiella species were correctly classified at the species level with 92% accuracy, classifying all three K. oxytoca isolates correctly. These findings should guide future applicative studies, increasing the scope of Raman spectroscopy's suitability for clinical applications.

Trends in pharmaceutical analysis and quality control by modern Raman spectroscopic techniques

Silge, A., Karina Weber, D. Cialla-May, L. Müller-Bötticher, D. Fischer, and J. Popp.
In: TrAC Trends in Analytical Chemistry, 116623 (12 April 2022)

The application of Raman spectroscopic methods in life science applications has entered a new era due to advances in instrumentation, miniaturization and, most importantly, increased interdisciplinary dialogue between spectroscopists and end users such as clinicians or pharmacists. Advances in laser sampling technologies, optical instruments and data analysis provide new possibilities for optical sensing and chemical imaging. Raman-based technologies are being driven by these new developments, opening new application for advanced pharmaceutical development and manufacturing. In addition, non-destructive and label free quality testing of drug formulations, Raman sensing is of particular interest for process-related aspects in bioprocessing. The trend to personalized medicine, particularly for pediatric and geriatric applications increased the number of prescriptions of individually tailored medicines manufactured in public pharmacies. This article discusses trends and updates of Raman based analytics and how Raman spectroscopic information can create value for modern pharmaceutical applications and processes. The first section reviews Raman modalities that have great potential for on-site applications in the pharmaceutical industry as well as in research and development. The second section provides an overview of innovative Raman spectroscopic devices and the many possibilities in terms of analytical capabilities and sample properties. Photonic data science has been an important driver for Raman based technologies breakthroughs in the last decade and help to standardize Raman spectral analysis, which is discussed in the third section. In the last section we discuss how to translate Raman-based innovations into user-friendly on-site applications.

Detection of multi-resistant clinical strains of E. coli with Raman spectroscopy

Amir Nakar, Aikaterini Pistiki, Oleg Ryabchykov, Thomas Bocklitz, Petra Rösch and Jürgen Popp
In: Analytical and Bioanalytical Chemistry (6 January 2022): 117973

In recent years, we have seen a steady rise in the prevalence of antibiotic-resistant bacteria. This creates many challenges in treating patients who carry these infections, as well as stopping and preventing outbreaks. Identifying these resistant bacteria is critical for treatment decisions and epidemiological studies. However, current methods for identification of resistance either require long cultivation steps or expensive reagents. Raman spectroscopy has been shown in the past to enable the rapid identification of bacterial strains from single cells and cultures. In this study, Raman spectroscopy was applied for the differentiation of resistant and sensitive strains of Escherichia coli. Our focus was on clinical multi-resistant (extended-spectrum β-lactam and carbapenem-resistant) bacteria from hospital patients. The spectra were collected using both UV resonance Raman spectroscopy in bulk and single-cell Raman microspectroscopy, without exposure to antibiotics. We found resistant strains have a higher nucleic acid/protein ratio, and used the spectra to train a machine learning model that differentiates resistant and sensitive strains. In addition, we applied a majority of voting system to both improve the accuracy of our models and make them more applicable for a clinical setting. This method could allow rapid and accurate identification of antibiotic resistant bacteria, and thus improve public health.

Noise Sources and Requirements for Confocal Raman Spectrometers in Biosensor Applications

Jahn, Izabella J., Alexej Grjasnow, Henry John, Karina Weber, Jürgen Popp, and Walter Hauswald.
In: Sensors 21, no. 15 (January 2021): 5067

Raman spectroscopy probes the biochemical composition of samples in a non-destructive, non-invasive and label-free fashion yielding specific information on a molecular level. Nevertheless, the Raman effect is very weak. The detection of all inelastically scattered photons with highest efficiency is therefore crucial as well as the identification of all noise sources present in the system. Here we provide a study for performance comparison and assessment of different spectrometers for confocal Raman spectroscopy in biosensor applications. A low-cost, home-built Raman spectrometer with a complementary metal-oxide-semiconductor (CMOS) camera, a middle price-class mini charge-coupled device (CCD) Raman spectrometer and a laboratory grade confocal Raman system with a deeply cooled CCD detector are compared. It is often overlooked that the sample itself is the most important “optical” component in a Raman spectrometer and its properties contribute most significantly to the signal-to-noise ratio. For this purpose, different representative samples: a crystalline silicon wafer, a polypropylene sample and E. coli bacteria were measured under similar conditions using the three confocal Raman spectrometers. We show that biosensor applications do not in every case profit from the most expensive equipment. Finally, a small Raman database of three different bacteria species is set up with the middle price-class mini CCD Raman spectrometer in order to demonstrate the potential of a compact setup for pathogen discrimination.

Bacterial Phenotype Dependency from CO2 Measured by Raman Spectroscopy

Wichmann, Christina, Thomas Bocklitz, Petra Rösch, and Jürgen Popp.
In: Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021): 119170.

In recent years, Raman spectroscopy has become an established method to study medical, biological or environmental samples. Since Raman spectroscopy is a phenotypic method, many parameters can influence the spectra. One of these parameters is the concentration of CO2, as this never remains stable in nature, but always adjusts itself in a dynamic equilibrium. So, it is obvious that the concentration of CO2 cannot be controlled but it might have a big impact on the bacteria and bacterial composition in medical samples. When using a phenotypic method like Raman spectroscopy it is also important to know the influence of CO2 to the dataset. To investigate the influence of CO2 towards Raman spectra we cultivated E. coli at different concentration of CO2 since this bacterium is able to switch metabolism from aerobic to microaerophilic conditions. After applying statistic methods small changes in the spectra became visible and it was even possible to observe the change of metabolism in this species according to the concentration of CO2.

Isolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy

Wichmann, Christina, Petra Rösch, and Jürgen Popp.
In: Analytical and Bioanalytical Chemistry 413, no. 20 (August 2021): 5193–5200

Raman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix. Graphical abstract.

Raman Stable Isotope Probing of Bacteria in Visible and Deep UV-Ranges

Azemtsop Matanfack, Georgette, Aikaterini Pistiki, Petra Rösch, and Jürgen Popp.
In: Life 11, no. 10 (October 2021): 1003

Raman stable isotope probing (Raman-SIP) is an excellent technique that can be used to access the overall metabolism of microorganisms. Recent studies have mainly used an excitation wavelength in the visible range to characterize isotopically labeled bacteria. In this work, we used UV resonance Raman spectroscopy (UVRR) to evaluate the spectral red-shifts caused by the uptake of isotopes (13C, 15N, 2H(D) and 18O) in E. coli cells. Moreover, we present a new approach based on the extraction of labeled DNA in combination with UVRR to identify metabolically active cells. The proof-of-principle study on E. coli revealed heterogeneities in the Raman features of both the bacterial cells and the extracted DNA after labeling with 13C, 15N, and D. The wavelength of choice for studying 18O- and deuterium-labeled cells is 532 nm is, while 13C-labeled cells can be investigated with visible and deep UV wavelengths. However, 15N-labeled cells are best studied at the excitation wavelength of 244 nm since nucleic acids are in resonance at this wavelength. These results highlight the potential of the presented approach to identify active bacterial cells. This work can serve as a basis for the development of new techniques for the rapid and efficient detection of active bacteria cells without the need for a cultivation step.