Stable Isotope Raman Microspectroscopy (SIRM) for Quantitative and Nondestructive 3D Analysis: Investigation of SIRM Potential for Applications in Environmental Analysis / STARAMM


  • Exploring the potential of stable isotope (resonance) Raman microspectroscopy and surface-enhanced Raman scattering for characterization of microorganisms at single the cell level
  • Quantitative analysis of different environmental samples by means of SIRM
  • Validation of SIRM data with results of well-established stable isotope-based techniques (isotope-ratio mass spectrometry, IRMS and nanoscale secondary ion mass spectrometry, NanoSIMS)

Methods of Approach

  • Stable isotope Raman microspectroscopy (SIRM)
  • Resonance Raman spectroscopy and SERS


Since the discovery of stable isotopes in the early 1920’s, stable isotope-based analytical methods are gaining more and more relevance in different scientific fields. In contrast to well-established techniques like Isotope Ratio (IRMS) and Isotopic Dilution Mass Spectrometry (IDMS), as well as Mass Spectrometry Imaging (MSI) methods (e.g. based on Secondary Ion Mass Spectrometry, SIMS) and Nuclear Magnetic Resonance Spectroscopy (13C-NMR, D-NMR), the Stable Isotope Raman Microspectroscopy (SIRM) opens the possibility to perform not only quantitative spatially-resolved, but also nondestructive depth-resolved analysis of various (in)organic and (micro)biological samples.

SIRM is a nondestructive analytical technique which provides characteristic fingerprint spectra of samples with the spatial and depth resolution of a confocal optical microscope. On the one hand, these spectra contain the information on stable isotope-marked substances and the amount of a marker; on the other hand, they provide information on the chemical composition and structure of a sample. The SIRM analysis requires no or limited sample preparation and can be performed in situ and in vivo without interference of water.

In this project the SIRM potential for quantitative nondestructive 2D and 3D environmental analysis is investigated. We perform studies on the quantification of the isotope ratio and the absolute concentration of stable isotope/stable isotope-containing substances by spatially- and, especially, depth-resolved studies. Of particular interest are the possibilities for combining SIRM with the resonance (R) effect and the surface-enhanced Raman scattering (SERS). The resulting SIRRM and SISERS techniques allow us to improve the sensitivity of the quantitative studies.

First results:

We explored the potential, the applicability, and the limitations of SIRM, resonance SIRM, and SIRM in combination with SERS for the characterization of single bacterial cells (see Figure Kubryk et al. 2015 Anal Chem).

Furthermore, in this project we studied the possibilities of SERS in combination with stable isotopes (13C- and 15N-labeled compounds) at the single cell level (E. coli) and to understand the origin of certain bands in the SERS spectra. The SERS analysis of bacteria cultivated with 12C-, 13C- and 15N-compounds showed a very good reproducibility. Notably, a very sharp marker band at 733 cm-1 for 12C-microorganisms could be found. This band is significantly red-shifted to 720 cm-1, to 717 cm-1 and to 707 cm-1 for 13C-, 15N- and for 13C15N-labeled bacterial cells, respectively. With the help of the different labels the pronounced SERS band at 733 cm-1 could successfully be assigned to adenine-related compounds.

Additionally, we examined the potential of SIRM for the evaluation of differently enriched 13C-labeled humic acids as model substances for soil organic matter (SOM). The SOM itself can be linked to the soil water holding capacity. Artificial humic acids (HA) with known isotopic compositions were synthesized and analyzed by means of SIRM. Results were verified against isotope ratio mass spectrometry (IRMS). The limit of quantification was 2.1 × 10−1 13C/Ctot for the total region and 3.2 × 10−2 13C/Ctot for a linear correlation up to 0.25 13C/Ctot. Complementary nanoscale secondary ion mass spectrometry (NanoSIMS) analysis indicated small-scale heterogeneity. Our study shows that SIRM is well-suited for the analysis of stable isotope-labeled HA.


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 (invited 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 2018, 16th Anniversary Issue, 410, 923-931,

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, 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.