Initially, much work went into set-up of equipment and data processing pipelines. Our set-up includes TERS set-ups with different scanning probe microscopy feedback (AFM, STM), different illumination and collection geometries (top / side / bottom), and an electrochemical TERS setup for performing experiments in liquid. We have also have environmental control for two of our TERS systems to reduce the contamination of the sample and degradation of the STM-TERS tip during the measurement. This has also helped in extending the lifetime of the TERS tip, hence we can perform a high-resolution TERS map without loss of enhancement of the tip.
In terms of sample preparation, the production of artificial membranes formed on a Langmuir-Blodgett trough and their transfer onto template stripped gold, Au (111) single crystals, mica, glass, and silicon for TERS measurement has been optimized. We have performed isotherm studies of the cationic antimicrobial peptide indolicidin and its interaction with DOPC. We have measured confocal Raman spectra of the peptide and identified the Raman bands of interest. Deposition of biomimetic and bacterial cell membranes (bilayers) on gold via vesicle fusion was optimized. We have achieved STM-TERS imaging of a DPPC self-assembled monolayer on gold, and are finishing a study on high-resolution chemical imaging of biphasic lipid systems consisting of DPPC, deuterated (d31) SM, and cholesterol.
For providing a better understanding of TER spectra, we simulate Raman spectra of molecular systems, allowing the assignment of the complex TERS and Raman spectra. We have written a robust software code for processing and analyzing the TERS data, and are continuously updating it, adding high-level data analysis routines to extract the maximum amount of information from our data. Furthermore, it also helps us in understanding and dealing with the large amount of data generated in our experiments.
For typical metal-organic coordination systems in biological samples, we achieved the molecular-scale chemical imaging of the molecular specificity, orientation, and spatial distribution of model coordination complexes using TERS. We were able to visualize the soft coordination species spectroscopically on the scale of a single molecule under ambient conditions, which has laid the foundation for further in-situ molecular-scale TERS imaging of the biological dynamics on surfaces.
For the investigation of soft samples like polymers and biological samples, we studied the correlation between TERS signal and AFM feedback mode in AFM-TERS experiments. Counterintuitively, tapping mode yielded a systematically higher Raman signal intensity, despite the weaker probe-sample interaction. We interpreted this result as a consequence of the lower molecular perturbation generated in tapping mode. Conversely, the strong probe-sample interaction in contact mode can alter the structure and orientation of the sample, leading to a lower signal. These findings are of key relevance for the development of TERS of delicate samples.
A lot of work was done to improve the performance of the bottom-illumination AFM-TERS system, starting with the instrument realignment up to designing a data processing software. This system was used in a comprehensive study of polystyrene (PS)-poly(methyl methacrylate) (PMMA) polymer blend film in which it is well known that the polymers phase separate laterally. However, we also detected a vertical phase separation of polymers within 20 nm from the sample surface, which was not detected before. The same AFM-TERS system was also used to study pancreatic cancer cells (BxPC3 cell line). High resolution of this instrument allowed us to reveal segregation of lipids and proteins and distinguish between cell domains rich in different proteins, which we couldn't detect using the confocal Raman spectroscopy. These studies demonstrate the potential of TERS to study cell walls under ambient conditions.
