Stable Isotope Raman Microspectroscopy (SIRM) for Quantitative and Nondestructive 2D & 3D Analysis of Biofilms: Microbial Degradation of Microplastic / StaR_Mic2


  • Exploring the potential of stable isotope (resonance) Raman microspectroscopy for characterization of biodegradation of microplastic on the single-cell level
  • Tracing deuterium from labeled plastic into microbial biomass
  • Comparison of SIRM data with results of well-established stable isotope-based techniques (laser absorption spectroscopy of D2O, fatty acid methyl ester gas chromatography mass spectrometry (FAME-GC-MS)

Methods of Approach

  • Stable isotope Raman microspectroscopy (SIRM)
  • Resonance Raman spectroscopy
  • Fatty acid methyl ester gas chromatography mass spectrometry (FAME-GC-MS)
  • Laser absorption spectroscopy


Stable isotope-based analytical methods gain increasing relevance in different scientific fields. Although mass spectrometry-based (MS) methods enable sensitive analysis of bulk samples (e.g., isotope ratio mass spectrometry, IRMS) or provide a spatial resolution down to 50 nm (e.g., nanoscale secondary ion mass spectrometry, NanoSIMS), these methods are destructive and require time-consuming sample preparation. Here, a combination of Raman microspectroscopy (RM) with the stable isotope approach – stable isotope Raman microspectroscopy (SIRM) – can extend the capabilities of the well-established techniques with a nondestructive, quantitative, and spatially-resolved analysis. SIRM provides characteristic fingerprint spectra of samples with the spatial resolution of a confocal optical microscope, containing information on stable isotope-labeled substances and the amount of a label (based on red shift of bands of the labeled substances). Simultaneously, these spectra deliver information on the chemical composition and structure of samples. Furthermore, this method requires no or limited sample preparation, and can be performed in situ and in vivo without spectral interference of water.

The feasibility of SIRM for the quantitative studies of different samples, incl. humic substances, microorganisms and biofilms, has been demonstrated during the first phase of the applicant project (STARAMM). SIRM has been also successfully combined with resonance and surface-enhanced Raman scattering (SERS) effects, to improve its sensitivity. The achieved results as well as current studies of other researchers indicate a high potential of SIRM for the characterization of microbial communities at the single cell level. This method provides information on the carbon metabolism / flow and the cell activity, and has already been tested for the analysis of the degradation of environmental pollutants. However, no research has been performed so far on the microbial degradation of a most prominent emerging pollutant in the aquatic environment – microplastic (MP).

The goal of this follow-up project is to develop and evaluate a SIRM-based method for quantitative and nondestructive analysis of bacteria involved in the biodegradation of MP. SIRM has a potential to provide novel information on the carbon and hydrogen assimilation and general metabolic activity of microorganisms from plastic-degrading bacteria on a single-cell level. As an alternative to expensive 13C-labelled substrates, the potential of deuterated polymers is investigated. The obtained SIRM results will be validated with FAME-GC-MS data to measure the deuterium uptake into microbial lipids as biomass markers and with laser absorption spectroscopy data to monitor the heavy water content in the aqueous medium as the final degradation product. Altogether, this should help us to establish a reliable method for the analysis of biodegradation of (micro)plastic in the aquatic environment, thus providing us with (direct) information on the single-cell level.


J. Weng, K. Müller, O. Morgaienko, M. Elsner, N. P. Ivleva, Multi-element Stable Isotope Raman Microspectroscopy of Bacterial Carotenoids Unravels Rare Signal Shift Patterns and Single-cell Phenotypic Heterogeneity, Analyst 2023, 148, 128–136,

R. Weiss, M. Palantinszky, M. Wagner, R. Niessner, M. Elsner, M. Seidel & N. P. Ivleva, Surface-Enhanced Raman Spectroscopy of Microorganisms: Limitations and Applicability on the Single-Cell Level. Analyst 2019, 144, 943-953,

N. P. Ivleva, P. Kubryk & R. Niessner, Raman Microspectroscopy, Surface-enhanced Raman Scattering Microspectroscopy, and Stable-isotope Raman Microspectroscopy for Biofilm Characterization. Analytical & Bioanalytical Chemistry 2017, 409, 4253-4375, review)

A. C. Wiesheu, R. Brejcha, C. W. Mueller, I. Kögel-Knabner, M. Elsner, R. Niessner & N. P. Ivleva, Stable-Isotope Raman Microspectroscopy for the Analysis of Soil Organic Matter. Analytical & Bioanalytical Chemistry 2017, DOI: 10.1007/s00216-017-0543-z 

P. Kubryk, Niessner & N. P. Ivleva, On the Origin of the Band at around 730 cm-1 in SERS Spectra of Bacteria: Stable Isotope Approach. Analyst 2016, 141, 874-2878

P. Kubryk, J. Kölschbach, S. Marozava, T. Lüders, R. Meckenstock, R. Niessner & N. P. Ivleva; Exploring the Potential of Stable Isotope (Resonance) Raman Microspectroscopy and SERS for the Analysis of Microorganisms at Single Cell Level. Analytical Chemistry 2015, 87, 6622-6630


N. P. Ivleva, Applicability of Raman Microspectroscopy for Environmental Analysis, Institute of Biogeochemistry and Pollutant Dynamics, Swiss Federal Institute of Technology (ETH) Zurich, 29.10.2019, Zürich, Switzerland

N. P. Ivleva, SERS in Combination with Stable Isotope Approach for Analysis of Bacteria at Single Cell Level, SciX 2018, 21.-26.10.2018, Atlanta, USA, (invited)

N. P. Ivleva, Stable Isotope Raman Microspectroscopy for Environmental Analysis: Feasibility Studies, Analytica conference 2018, New Developments in Analytical Spectroscopy – The Power of Light, 10.-13.4.2018, Munich, Germany (invited)

R. Weiss, P. Kubryk, M. Seidel, R, Niessner, M. Elsner & N. P. Ivleva, Applicability of SERS in Combination with Stable Isotope Approachfor Characterization of Microorganisms at Single Cell Level, FT-IR Spectroscopy in Microbiological and Medical Diagnostics 2017, 19.-20. October 2017, RKI, Berlin, Germany

N. P Ivleva, P. Kubryk, R. Weiss, Michael Seidel, Reinhard Niessner, SERS in Combination with Stable Isotope Approach for Characterization of Bacteria at Single Cell Level, International Conference on Enhanced Spectroscopies (ICES), 4.-7. September 2017, Munich, Germany

N. P. Ivleva, Raman Microspectroscopy for Nondestructive 2D and 3D Analysis of Environmental Samples, Department of Bioengineering and Electrical and Computer Engineering, Northeastern University, 09.05.2017, Boston, USA (invited lecture)

N. P. Ivleva, A. C. Wiesheu, R. Weiss, P. Kubryk, R. Brechja, M. Elsner, C. W. Müller, I. Kögel-Knabner, Reinhard Niessner,  On the Potential of Stable Isotope Raman Microspectroscopy (SIRM) for Nondestructive Spatially-resolved Analysis of Environmental Samples, ANAKON 2017, 3.-6. April 2017, Tübingen, Deutschland

N. P. Ivleva, Raman Microspectroscopy and SERS for Analysis of Biofilms, Hong Kong University of Science and Technology, HKUST, 8.6.2016, Hong Kong, China (invited lecture)

P. Kubryk, J. S. Kölschbach, S. Marozava, T. Lueders, R. U. Meckenstock, R. Niessner & N. P. Ivleva, Stable isotope (Resonance) Raman Microspectroscopic and SERS Analysis of Single Microbial Cells, FT-IR Spectroscopy in Microbiological and Medical Diagnostics 2015, 15.-16.10.2015, RKI, Berlin, Germany

N. P. Ivleva, P. Kubryk, A. C. Wiesheu & R. Niessner, Nondestructive Quantitative Analysis by Stable Isotope Raman Microspectroscopy (SIRM): Applications in Environmental Analysis, ANAKON2015, 23.-26. March 2015, Graz, Austria


R. Weiß, R. Nießner, M. Seidel, N. P. Ivleva, Auswirkung physiologischer Bedingungen auf Detektion von Mikroorganismen mittels Oberflächen-verstärkter Raman-Spektroskopie, WASSER 2018, 07.-09.05.2018, Papenburg, Deutschland.

R. Weiß, R. Nießner, M. Seidel, N. P. Ivleva, Raman-Mikrospektroskopie für zerstörungsfreie, dreidimensionale Analysen von Biofilmen, ANAKON 2017, 03.-06.04.2017, Tübingen, Deutschland.

A. C. Wiesheu, R. Brejcha, M. Elsner,  R. Nießner, N. P. Ivleva, Organische Substanzen im Boden zur Erhöhung der Wasserrückhaltefähigkeit: Analyse mittels Stabilisotopen-Raman-Mikrospektroskopie, WASSER 2017, 22.-24.05.2017, Donaueschingen, Deutschland.

P. Kubryk, R. Weiss, R. Niessner, N. P. Ivleva, Stable-isotope SERS analysis of microorganisms in aqueous systems, WASSER 2016, 02.-04.05.2016, Bamberg, Deutschland.

A. C. Wiesheu, R. Nießner, N. P. Ivleva, Stabilisotopen-Raman-Mikrospektroskopie (SIRM) zur Analyse von organischen Substanzen im Boden, WASSER 2016, 02.-04.05.2016, Bamberg, Deutschland.