A key utility of fluorophores lies in sensing applications: the detection of changes to emission caused by differences in their microenvironment. The rational design of fluorescent sensors remains a significant challenge because of the complexity of factors which control molecular deactivation pathways. Here, in an effort to define the structural criteria underlying the fluorescence turn-on response of Hoechst 33258 (H33258) upon binding to the DNA minor groove, we examine this sensor's intrinsic properties in minimalist microenvironments. We first characterised the intrinsic photophysics of gaseous mono- and di-protonated H33258 ions, then introduced intermolecular interactions by complexation with double-stranded (ds) DNA. Selected-ion laser-induced fluorescence (SILIF) and photodissociation of the gaseous monoprotomers indicate the presence of multiple populations with distinct fluorescence and dissociation properties. We assign one of these to a kinetically-trapped form which is protonated at the site favored in solution. The other form exhibits a more intense emission band which is shifted by more than 6000 cm-1 to the red of the first form. Quantum chemical calculations reveal that this second population is likely a newly-identified protomer, which is considerably more stable in the gas phase than conformations with the solution protonation site. Two routes that increase the fluorescence of H33258 in solution - formation of the diprotomer and complexation with dsDNA - do not produce an increase in fluorescence in the gas phase. However, two other outcomes parallel behaviour. First, the similarity of action spectra of the gaseous dsDNA-H33258 complex and the unbound diprotomer suggest that the dye may be diprotomeric when in complex with gaseous dsDNA. Second, the photodissociation power dependence measurements indicate the presence of at least two distinct populations of both H33258 in complex with dsDNA and in its unbound diprotomeric form. Overall, the results reported here reveal unexplored aspects of the potential energy landscape of H33258, including a new, stable, highly-fluorescent form that may be useful to consider in sensing applications. Moreover, the results reinforce how structure, deactivation pathways and other photophysical properties are intertwined for this DNA-binding dye, which may offer strategies for improved control of DNA-targeting drugs and sensors.