Fluorescence spectroscopy provides impressive insights into biological systems – and the dynamics of living cells in particular – at high spatial and temporal resolutions. Techniques such as STED, PALM and STORM have been improving spatial resolution in recent years, pushing it down to the low-nanometer range. This breakthrough was recognized in 2014 with the Nobel Prize in Chemistry. As a rule, fluorescence spectroscopy requires a “luminous” label, which is attached to the molecule to be investigated. One such label is green fluorescent protein (GFP), for example. Indeed, the Nobel Prize in Chemistry was awarded (in 2008) for the discovery and application of GFP, which underlines the momentous importance of suitable labels for fluorescence spectroscopy. Yet the labeling technique is not without its difficulties: the tagging process can be complex, the label itself is liable to bleaching and it may even distort the process to be investigated.
There is where label-free vibrational spectroscopy can offer a complementary method. A vibrational spectrum is generated by the intrinsic vibration of a molecule. This enables a molecule to be identified directly by means of its spectrum. This vibration spectrum can be measured either in absorption with IR spectroscopy or in emission with Raman spectroscopy. In chemistry, IR and Raman spectroscopy have been deployed routinely for many decades now. In comparison to NMR spectroscopy or X-ray structure analysis, however, both are considered to be low-resolution methods in routine analysis. Systematic improvements have meant that vibrational spectroscopy methods are now enjoying renewed popularity in research, especially in the last few years.
Impressive advances in the field of vibrational spectroscopy in particular were presented in 2015 at the 8th International Conference on Advanced Vibrational Spectroscopy (ICAVS, http://icavs8.icavs.org/) held at TU Vienna and at the 16th European Conference on the Spectroscopy of Biological Molecules (ECSBM, http:// www.ecsbm2015.de/) held at Ruhr University Bochum. These conferences impressively demonstrated how the use of FTIR difference spectroscopy was able to determine time-resolved molecular reaction mechanisms of proteins and protein interactions at the atomic level. With difference spectroscopy, only the amino acids involved in the protein reaction are selected from the background absorbance of the entire protein. In contrast to routine analysis, this achieves a very high level of resolution. As one example of current work, the molecular mechanism of channelrhodopsins is being investigated in detail with this approach. Channelrhodopsins are key tools in optogenetics. The deployment of photolabile substances such as caged GTP or ATP permits the detailed investigation of GTPases and ATPases. One example that can be mentioned here is the Ras protein, whose variants, created by oncogenic mutations, are a major driver in carcinogenesis.
Particularly impressive, however, is the label-free imaging of tissues and cells developed in recent years. For the imaging of individual cells, Raman spectroscopy is primarily deployed, while tissue imaging predominantly utilizes FTIR spectroscopy. The spatially-resolved vibration spectra measured facilitate the label-free classification of cell organelles or tissue components. Since tissue annotation is label-free, the method can be automated. By using FTIR imaging, cancer tissue in particular can be identified with a sensitivity and specificity of over 95 %, as has been demonstrated for prostate, lung, bladder and colon tissue, for example. Moreover, these techniques not only permit the identification of cancer tissue but also facilitate predictive annotations – such as the subclassification of pulmonary adenocarcinomas, for example. They therefore supply an important basis for treatment decisions made by the attending physician. Raman spectroscopy can do more than merely characterize cells, however: it can also be used to analyze the pharmacokinetics of therapeutically effective small molecules.
Over the next few years, the use of quantum cascade lasers in IR spectroscopy and non-linear techniques such as CARS in Raman spectroscopy means substantial progress is to be expected for their clinical application in particular, since these advances will permit subclassifications to be made with much greater speed and higher precision. These subclassifications are decisive for differential diagnosis. Research scientists have been pioneering advances in time-resolved FTIR spectroscopy and vibrational imaging. For the future, is it important not only to support this field with funding but to bring manufacturers on board with the aim of applying the considerable potential of label-free methods to translational clinical applications. A precise diagnosis is the prerequisite for personalized therapy.