Fraunhofer Institute for Interfacial Engineering and Biotechnology
At the Fraunhofer IGB we work on the application of Raman spectroscopy for biological tissues. Our goal here is to develop a method for the non-invasive sterility control of transplants and non-invasive and marker-free examination of single cells.
Fields of research
Raman spectroscopy uses the interaction of mochromatic light such as laser light and the sample under observation, hereby offering the possibility of a non-destructive and label-free characterization of biological material. Such a method could set new standards in regenerative medicine. It would offer a completely new and in addition lower-priced quality control for the individualized therapy.
For biological applications such as the investigation of microorganisms and single cells, we have combined the Raman spectrometer with a light microscope. The advantage of this technology in relation to comparable methods as for example infrared spectroscopy, is in the possibility of working in aqueous solutions. This makes Raman spectroscopy one of the most suitable techniques for carrying out analyses directly in cell culture media and without complicated sample preparation.
Our focus is to find the relevant information in the sum of the acquired data and to establish a technique for the application in tissue engineering and regenerative medicine.
An important criterion for the quality control of cells in regenerative medicine is the monitoring of the differentiation path of different cell types and the determination of cell viability and differentiation state. While observing these parameters, important steps for the production of tissue-engineered products can be determined. In our studies, we have shown that Raman spectroscopy can be used to categorize a variety of cell types. This gives us the ability to create pure cell cultures. Additionally, the validation of cell viability is an important and recurring parameter in quality control. In this area, we were able to identify spectral regions that allow for the classification of viable, necrotic and apoptotic cells. In our current studies, we established standard protocols to monitor the directed differentiation of pluripotent stem cells.
In addition to the analysis of isolated cells, Raman spectroscopy is a suitable method for the analysis of cells within their natural three-dimensional (3D) environment. In previous studies, we successfully identify and characterize skin cells, such as keratinocytes, melanocytes and fibroblasts, within biopsies. In addition, we could analyze cells within a 3D in vitro skin model without the need for histology and immunohistochemistry. Furthermore, we demonstrated that Raman spectroscopy could make a qualitative distinction between native and pathological tissue matrix, which will enable the ability to diagnose transplants pre-implantation.
Currently, we are optimizing and automating the individual steps of the measurement process, such as image analysis. Additionally and in close collaboration with the University Hospital Tübingen, we are building a spectrum database of the different cells and tissues we have analyzed.
Good to know
The principle of Raman spectroscopy
The marker-free and non-invasive analysis by means of Raman spectroscopy is based on an effect discovered by the Indian Nobel Laureate C. V. Raman in 1928. As a result of the interaction of incident laser light on molecules in a specimen, a frequency shift of the scattered backlight occurs, which can be detected as a Raman spectrum. For each individual molecule of a specimen characteristic patterns are thus shown, similar to a fingerprint.
Raman spectroscopy enables the chemical analysis of samples with high spatial resolution of less than 1 µm. The method is used today in a wide field of research, particularly for material characterization, for example by semiconductors or chemicals, and also for investigations of gases and exhaust gases.
Raman spectroscopy for biological applications
For biological applications such as the investigation of microorganisms and single cells, the Raman spectrometer can be combined with a light microscope. The advantage of this technology in relation to comparable methods as for example infrared spectroscopy, is in the possibility of working in aqueous solutions. This makes Raman spectroscopy one of the most suitable techniques for carrying out analyses directly in cell culture media and without complicated sample preparation.
- "BioRaman" project – On-line quality control for accelerating drug development and individualized therapy using Raman spectroscopy
In the Fraunhofer project "BioRaman" in co-operation with the Fraunhofer Institutes IPM and IBMT the Fraunhofer IGB has developed a device and method for non-destructive and label-free characterization of biological material by Raman spectroscopy. Within the project we also developed a flow cell for particle positioning (patented).
- "Marker-free and chemically selective microscopy for rapid cell screening"
In this project funded by the Baden-Württemberg Foundation, we study the marker-free analysis of mesenchymal stem cells and their differentiation into various cell lines using Raman spectroscopy and Coherent Anti-Stokes Raman spectroscopy (CARS). The focus is on the detectability of cellular differentiation using the above techniques.
- Verification of cellular metabolic activities
In another, Fraunhofer-funded project, we have examined the metabolic changes of algal cells, in terms of their bio-based production of omega-3 fatty acids, beta-interferon and factor VII, which is a clotting factor. It was found that the metabolic shift between producing and non-producing cells could be identified using Raman spectroscopy. This demonstrates the high sensitivity of the method and opens new avenues of application of Raman spectroscopy for quality control.
- Studies and testings
- Histology, analysis of tissues and supernatants
- Active agent efficacy detection on single cell level (prospective)
- Characterization of carrier materials
- Cell characterization and sorting
- Cell isolation from biopsy material
- Microscopy (AxioVert 200M, Zeiss)
- Micro-dissection (MicroBeam, PALM-Zeiss)
- Laser scanning technology
- Raman spectrometer
- Real-time PCR
- Flow cytometry
Raman spectrometer with fluorescence microscope
At the Fraunhofer IGB, we have a unit for the investigation of biological and medical material, which is equipped with a 785 nm diode laser (NIR near infra-red laser) and is coupled to a fluorescence microscope. This reduces the risk of damaging the specimen by applying higher energy wavelengths. This Raman spectrometer enables the investigation and the direct comparison of fluorescence-marked samples with the associated Raman spectra.
Together with a patented flow cell for particle positioning on a microporous membrane for filtering and concentrating the sample, even the smallest of particles (microorganisms, single human cells) can be investigated. The system was developed in cooperation with the Fraunhofer Institutes for Physical Measurement Techniques IPM and for Biomedical Engineering IBMT.
- Votteler, M. et al. (2012) Raman spectroscopy enables the non-contact, marker-free monitoring of cells and extracellular matrix. J Vis Exp in press
- Votteler, M. et al. (2012), J Biophotonics 5(1): 47–56
- Pudlas, M. et al. (2011), Tissue Eng Part C Methods 17(10): 1027-1040
- Pudlas, M. et al. (2011), Medical Laser Application 26(3): 119-125
- Pudlas M, Koch S, Bolwien C, Walles H, Raman spectroscopy as a tool for quality and sterility analysis for tissue engineering applications like cartilage transplants, Int J Arti Organs, 33(3), 228-237, 2010.
- Koch S, Dreiling M, Gutekunst M, Bolwien C, Thielecke H, Mertsching H, Discrimination of microorganisms and cells in tissue engineering by Raman spectroscopy, Proceedings of SPIE, 7368-46, 2009.
- Mertsching H, Sulz G, Thielecke H, Bolwien C, Koch S, Vorrichtung und Verfahren zur Analyse biologischer Proben, Patentschrift, Deutsches Patent- und Markenamt, DE 10 2006 053 540 B3, Tag der Veröffentlichung 31.01.2008.
- Bolwien C, Sulz G, Becker S, Thielecke H, Mertsching H, Koch S, Rapid Detection of Bacterial Contamination in Cell or Tissue Cultures Based on Raman Spectroscopy, Proceedings of SPIE, Vol. 6853, 68530F, 2008.