Book excerpt: While ions are held in the volume of a quadrupole ion trap mass spectrometer it is possible to use gas-phase optical spectroscopy to extract intrinsic information from the trapped species. This information is unencumbered by influences and interferences that populate the condensed phase. Under these conditions, it is possible to develop an understanding of the intersection of the intra- and intermolecular influences on a molecule. Doing so by beginning at the baseline of a bare ion and building up complexity in a controlled and calculated fashion. This research aims to fill in existing knowledge gaps by monitoring the effect of discrete changes on trapped ions. Chapters 2 and 3 of this thesis focus on examining intrinsic photophysical properties from groups of common fluorescent dyes (oxazines and rhodamines). The subtle structural change of the deuteration exchange of labile hydrogens resulted in significant changes in the measured fluorescence of oxazines but minimally affected structurally related rhodamine dyes. These may function as benchmark results and enable a better understanding of the interplay between structure and quantum efficiency. In combination with computation, the new insights promise to aid the effective and deliberate design of new fluorescent probes. The fourth chapter of this thesis describes the effects of increasing the internal energy of interrogated systems; via the design, implementation, and application of an intra-vacuum heating system. Effects were monitored using optical probing in combination with heating for several systems. These effects were varied, from the subtle shifting of rhodamine emission spectra to a rejection of the gas phase effectiveness of fluorescence thermometer molecule rhodamine B, and inducing a FRET monitored conformational change in a mutant variant of ubiquitin. Using this method of purposefully added complexity (deuteration and increased internal energy) it is possible to better understand how and why these effects occur. The work in these chapters represents essential steps in taking mass spectrometry from an important tool for sensitive and selective detection to an expanded role as an important investigative space to determine the nature of the molecules we use every day.