This dissertation describes the design, synthesis, and applications of photoactivatable fluorescent probes in the visible light region. Visible light-activated fluorescent probes are highly desirable due to the spatiotemporal control and widely available light sources. The use of photoactivatable probes in biological systems is optimal as the activation method is straightforward and non-invasive. A single atom substitution in fluorescent dyes has been demonstrated to efficiently generate lightactivatable fluorogenic cages. This methodology has been developed for use in superresolution imaging, photodynamic therapy, and inorganic mercury detection. The development of fluorogenic tetrazine probes allows the design of probes that are not limited to a single fluorophore core; it was then demonstrated that visible light could cleave the tetrazine moiety in fluorophore-conjugated dyes. The introduction of 3- methyilthio tetrazines improved turn-on efficiency and redshifted the wavelength of dissociation. The organization of this thesis is as follows: Chapter 1 will introduce photoremovable protecting groups ranging from UV to far red. The applications of caged groups are discussed in the fields of synthesis and biology. Chapter 2 describes the use of s-tetrazines as photocages. Light-activated fluorescence affords a powerful tool for monitoring subcellular structures and dynamics with enhanced temporal and spatial control of the fluorescence signal. Here, we demonstrate a general and straightforward strategy for using a tetrazine phototrigger to design photoactivatable fluorophores that emit across the visible spectrum. Tetrazine is known to efficiently quench the fluorescence of various fluorophores via a mechanism referred to as through-bond energy transfer. Upon light irradiation, restricted tetrazine moieties undergo a photolysis reaction that generates two nitriles and molecular nitrogen, thus restoring the fluorescence of fluorophores. Significantly, we find that this strategy can be successfully translated and generalized to a wide range of fluorophore scaffolds. Based on these results, we have used this mechanism to design photoactivatable fluorophores targeting cellular organelles and proteins. Compared to widely used phototriggers (e.g., o-nitrobenzyl and nitrophenethyl groups), this study affords a new photoactivation mechanism, in which the quencher is photodecomposed to restore the fluorescence upon light irradiation. Because of the exclusive use of tetrazine as a photoquencher in the design of fluorogenic probes, we anticipate that our current study will significantly facilitate the development of novel photoactivatable fluorophores. Chapter 3 provides further insight in the development of tetrazine photocages. The photolysis rates of carbon and sulfur tetrazines were obtained, showing a redshift in the photolysis wavelength when sulfur substitution is incorporated. This strategy was further implemented in through bond energy transfer tetrazine cassettes, with a one-pot methodology to convert carboxylic esters into 3-methylthio tetrazines. Higher turn-on ratios were obtained compared to 3-methyl capped tetrazine fluorophore counterparts. This strategy allowed the use of green light to unmask BODIPY based dyes. Chapter 4 will introduce the design and synthesis of a single-atom fluorescence switch. Applications of this methodology in cell imaging and photodynamic therapy are discussed. Chapter 5 of this thesis will detail the application of thionated fluorophores in the detection of inorganic mercury. By performing a single-atom replacement within common fluorophores, we have developed a facile and general strategy to prepare a broad-spectrum class of colorimetric and fluorogenic probes for the selective detection of mercury ions in aqueous environments. Thionation of carbonyl groups from existing fluorophore cores results in a great reduction of fluorescence. In the presence of mercury ions, the resulting thiocaged probes are efficiently desulfurized to their oxo derivatives, leading to pronounced changes in chromogenic and fluorogenic signals. Because these probes exhibit high selectivity, excellent sensitivity, good membrane-permeability, and rapid responses towards mercury ions, they are suitable for visualization of mercury in both aqueous and intracellular environments.