In this thesis, the advanced spectroscopic techniques of atomic force microscopy - infrared (AFM-IR) spectroscopy and sum-frequency generation (SFG) spectroscopy are explored in detail for their innate ability to unravel the complexities of biological interfaces. The benefits of both techniques are demonstrated through applications to a variety of different interfaces, ranging from whole cells and aggregates (red blood cells and hair fibres) to artificial mimics (hydrogels) and thin molecular films (phospholipid monolayers). Specifically, the lateral chemical resolution and surface sensitivity of AFM-IR are used to chemically characterise the constituent components of biologically relevant systems below the diffraction limit whilst correlating these measurements to the nanoscale physical properties of the surface, such as topography, friction, and adhesion, that are accessible through traditional AFM. Similarly, the inherent surface specificity and sub-monolayer sensitivity of SFG is utilised, along with its ability to elucidate chemical structure and conformation, to probe oxidation in cell membranes, both in whole cells and monolayer mimics, thereby yielding a much deeper understanding of the oxidation mechanism due to reactive oxygen species (ROS) in physiological systems.

Furthermore, some limitations and challenges associated with the use of these techniques in biologically relevant systems are identified, characterised and discussed, along with potential ways to circumvent them or minimise their impact in such investigations. Specifically, the inability of AFM-IR to probe thick substrates under aqueous conditions is discussed due to the relevance to studying many biological systems under physiological conditions. A novel method of sample preparation is proposed to maintain pseudo-aqueous conditions for the substrate whilst avoiding the limitations associated with AFM-IR measurement. Additionally, the common application of SFG to study surfactant monolayers at the air-water interface, particularly phospholipids due to them modelling cell membranes, is critically assessed for some inherent issues. The first example of which is associated with the inherent fluidity of the monolayer that makes it highly susceptible to local heating from the necessarily large incident fields. The effects of this local heating are thoroughly investigated and modelled, leading to conclusions about how to minimise this disruption in future investigations. Finally, SFG simulations are then used to quantitatively assess the validity of a common assumption associated with SFG investigations of phospholipids, namely that both alkyl chains yield the same spectral contributions despite having different orientational distributions.