Raman Spectroscopy and Imaging in Bioanalytics

Since the discovery of the inelastic scattering of light, i. e. , the so-called Raman effect, (1) Raman spectroscopy has become an attractive tool in a high number of research fields including biology, chemistry, and medicine. The use of lasers as an excitation source and the combination of Raman spectroscopy with microscopes have dramatically increased the applicability for bioanalytical questions and research tasks since the 1970s, which has been accompanied by the system development and product launch of Raman microspectroscopic devices. (2) In particular, the Raman spectroscopic investigation of the protein lysozyme nicely demonstrates the development of this technique in bioanalytics. (3) In 1958, the first Raman spectrum of the biopolymer lysozyme was published showing 14 faint lines, recorded by using a mercury source excitation. Ten years later, 21 modes were identified using laser Raman spectroscopy. In 1970, a study was published to assign and interpret the origin of the modes in the Raman spectrum of native lysozyme by comparison with spectra obtained from mixtures of the constituent amino acids. (4) Since the early development of laser-excited Raman spectroscopy, this technique and its variants have proven to be a powerful tool in bioanalytics. Initially, pure substances and biomolecules were investigated. Step by step, the samples studied gained complexity, achieving Raman spectroscopic characterization of cells and tissue, identification of bacteria on the strain level and tumor margins, and detection of low molecular weight substances in body fluids. aim of this Review is to provide the interested reader detailed information and necessary knowledge to decide on the most promising Raman-based analytical detection scheme for the specific planned application scenario. Thus, we address young researchers and beginners in the field approaching the topic of Raman spectroscopy and encourage the pursuit of Raman-based techniques in bioanalytics. As not every spontaneous and coherent Raman-based technique combines all advantages in a single method, the researcher must decide on the most appropriate analytical approach to address the specific analytical question. Therefore, in Raman Spectroscopy and Related Techniques, the theoretical background of Raman spectroscopy and related techniques is provided. , physics, materials sciences, mineralogy, art and archeology, biology, biochemistry, and pharmacy as well as biomedicine are mentioned as applied fields of Raman spectroscopic approaches, which illustrates the huge potential of this method in (bio) analytics. (5) To understand the development of diseases, Raman spectroscopy and its variants are applied in cancer diagnosis to detect tumor markers, to investigate body fluids, to characterize cell lines, and to monitor tissue samples. (6) This provides molecular information that allows one to correctly identify tumor margins in tissue or a cancer noninvasively. In addition to the development of powerful and portable Raman spectrometers suitable for use in a clinical environment, the generation of comprehensive databases in combination with data analysis tools is important for a successful translation of Raman spectroscopic approaches into clinics. In the case of cardiovascular diseases, Raman-based techniques are used to detect biomarkers as well as to investigate cells and tissues up to the organ level, i. e. , the heart. (7) As another example of an application, Raman spectroscopy and its variants are used in the study and diagnosis of neurodegenerative diseases, which are associated with conformational changes, as Raman spectroscopy is sensitive to such structural changes and post-translational modifications of proteins. (8) Raman spectroscopy further demonstrated its potential in the field of malaria. (9) Here, monitoring biological processes of the parasite and the interaction with antimalarial drugs is summarized. Vibrational spectroscopic methods have gained importance for the analysis of body fluids such as urine, tear fluid, blood, or saliva. However, as those body fluids have a mixed composition, standardized sample collection and pretreatment protocols as well as detection procedures have to be developed to enable the translation into clinical practice. (10) Surface enhanced Raman spectroscopy (SERS) is known as a powerful analytical method to investigate and identify bacteria in clinical biochemistry. (11) Other popular and promising Raman-based approaches are the determination of the antioxidant status in humans by resonance Raman (RR) spectroscopy of carotene in skin, (12) which has already been commercialized as the Pharmanex BioPhotonic Scanner in 2003, and the near real-time intraoperative assessment of brain tumors by stimulated Raman scattering (SRS) microscopy, where convolution neural networks have already been trained on over 2. 5 million images. (13) We summarize the application in bioanalytics starting from low molecular weight substances followed by biomacromolecules, viruses, bacteria, and cells up to tissue and organs in Application in Bioanalytics. It is shown that a large number of bioanalytical tasks and questions can be addressed with Raman-based techniques, as they provide molecular and chemical structural information at different levels of lateral resolution and sensitivity. discussed applied fields of Raman spectroscopy and its variants have the following in common: a database in combination with suitable data analysis tools is of great importance to ensure a translation into clinical application in the future. (14) Therefore, in Data Analysis Concepts in Raman-Based Methods in Biophotonics, the data analysis concepts of Raman-based methods will be introduced. Finally, the Conclusion and Outlook will summarize the discussion and will provide an outlook.