1. Developing Confocal Raman-AFM and Fluorescence-AFM Imaging Techniques to Visualize Drug-Cell Interactions with Further Implications in Cellular Pathology Research Erhan Süleymanoğlu On leave from Upper Austrian Research GmbH Hafenstraße 47-51, 4020 Linz, Austria erhans@mail.ru OBJECTIVES: To assess OBJECTIVES: To assess major cellular events requires us to determine biomolecular structures at high resolution. These can range from 0.1 nm to 20 nm, which represents a gap between FRET and optical microscopy. Our research efforts are directed towards developing new methods for deciphering intra-molecular distance and force measurements in cells and tissues with relevance to cellular pathology. METHODS: METHODS: To achive this, our studies have started with the use of Confocal Raman Microscopy, which can be upgraded to Atomic Force Microscopy (AFM) or vice versa. This enable nondestructive sample analysis on the nanometer scale involving only minimal sample preparation. Afterwards, simultaneous combination of AFM and Fluorescence Microscopy (FM) was applied. The two types of microscopies individually provide unique information about cellular samples under study. Using these microscopy techniques simultaneously provides the user with high resolution imaging on specific molecules. CONCLUSIONS: CONCLUSIONS: In principle, such nanoscale distance and force measurements could be applied to tissue sections as tumor biopsies or to single cells. A single molecule imaging of this sort could classify patients according to drug-cancer cell interactions with the aim of choosing the most appropriate therapy for the individual tumor type. RESULTS: The Signal Transduction Cascade Scanning AFM/Confocal/Raman/Fluorescence system for Raman/Fluorescence and AFM/Raman (TERS) imaging Major Setup of AFM-Raman Configurations for Fluorescence-Raman (A) optical phase contrast, (B) fluorescence, (C) AFM, and (D) combined AFM/fluorescence of fixed human lung fibroblasts, 60µm scan. Combinatory fluorescence-AFM on bioconjugated protein-quantum dot (QD) system. (A) Registration of raw QD fluorescence signals (yellow-red) with AFM topography (grey scale) of the same sample area (8 × 8 μm2). The fluorescence signals were fit by 2D Gaussians to determine their centers with nanometer accuracy, a technique also known as fluorescence imaging with one nanometer accuracy (FIONA). In (B), FIONA signals are shown in red color, indicating localization probability of the fluorescence centers. The red box in (B) indicates the QD-protein-DNA complex shown magnified in (C and D) as top view and 3D representation, respectively. The scale bar in (D) corresponds to 30 nm. These zoom in figures demonstrate good FIONA-AFM overlay accuracy, allowing the identification of a fluorescently tagged molecule in the AFM topography from its fluorescence signal. (E) Schematic of Fluorescence-AFM set-up. The sample is deposited on a mica substrate (inset zoom, not to scale), excited from below by total internal reflection (TIR) fluorescence (black arrows) and mechanically scanned from above by the AFM. Excited fluorescence (grey arrows) is filtered through a narrow bandwidth emission filter and recorded by a CCD camera attached to the microscope tri-occular port behind a 1× to 4× pre-magnifier. In situ confocal Raman spectroscopic characterization of protein conformation