The chemistry behind formation of molecules is the basis for most of the optical spectroscopy, fluorescence microscopy and optical imaging. This involves electronic and vibronic states of molecules that gives rise to specific spectra as its fingerprint. So, the structure of the molecule along with the transitions involving electronic states plays crucial role. This leads to an important class of theory for approximate understanding of molecules called molecular orbital theory (MOT). In this chapter, we will discuss the formation of diatomic and polyatomic molecules along with the selection rules involving electronic states that gives rise to specific spectra including the phenomena of fluorescence. All this will have its base in MOT. Specifically, we will focus on fluorescence and laws governing them including Kasha’s law, Stokes shift and Frank-Condon principle. Then we will focus on a few well-known fluorescent dyes/proteins that are frequently used in fluorescence microscopy, and discuss their spectral properties based on MOT. Specifically, here we concentrate on photoactivable fluorophores that are key to super-resolution microscopy and spectroscopy.