Development of nanoscale infrared spectroscopic imaging for measuring cellular ultrastructure - ABSTRACT Electron and fluorescence microscopy now provide exquisite structural (~ nm) information using specific, labeled molecular species. However, only a small fraction of cellular species can be illuminated in any experiment and labeling techniques work primarily for large molecules and assemblies. Small molecules, such as metabolites, remain largely hidden without significant effort (e.g., single cell mass spectrometry). Molecular vibrational spectroscopies (infrared and Raman) offer a unique opportunity to study all molecules of life directly but are typically limited in the far field by optical resolution, suffer from low sensitivity when sampling volumes are reduced to nm scale or do not yet offer consistent, reproducible data for near-field imaging. Atomic Force Microscopy (AFM) combined with Infrared (IR) spectroscopy provides a powerful tool for label-free chemical imaging with nanoscale resolution. There were three main challenges that limited AFM-IR performance: (a) mechanical artifacts coupled the morphology and chemical domains, preventing chemical analyses, (b) high noise reduced analytical accuracy and precision and (c) low sensitivity precluded quantification of multiple species. Consequently, recorded data of biological materials were restricted to studies using point spectra to avoid image artifacts, used thick samples (~micron scale) for sufficient sensitivity or examined well-described and simple domains like amyloid deposits. Recent scientific advances have paved a path to overcome these limitations; however, legacy instruments cannot take advantage of these advances. Hence, in this proposal we will develop a new AFM-IR technology, dubbed resonance enhance null-deflection IR (RENDIR) spectroscopic imaging. RENDIR will include custom electronics for controlling the instrument based on our recent closed loop null deflection technique, optimized data recording using theoretical understanding of the image formation process, an optimized optical train with expanded range quantum cascade lasers, and novel optics integrated into a custom designed AFM. These advances will enable high-quality AFM-IR signals for all types of samples and by users who are not necessarily doctoral level experts. This innovative technology will be tested using gold standard samples and its limits characterized using fabricated polymeric samples of known composition and geometry as well as biomedical samples. Lithographically patterned polymer samples will first be used to validate both spectral and spatial performance. An isogenic cancer progression cell line will be used as a biological model, wherein all subcellular domains and local metabolites in cytoplasmic regions will be identified. The end of this project period will yield an AFM-IR measurement technology (RENDIR) that significantly surpasses state of the art by being free of mechanical artifacts, allowing ~5 nm (~64-fold smaller pixels) resolution, images with ~25x lower noise (due to null deflection), ~19x higher sensitivity (due to resonance enhancement), and ~50x higher data acquisition speed (real-time controls) as well as a quantitative concordance with gold-standard Fourier transform IR (FT-IR) spectra.
