Photochromic Compounds for Fluorescence Nanoscopy

Diffraction prevents the spatial resolution of fluorescent species co-localized within areas of nanoscaled dimensions. As a result, conventional fluorescence microscopes cannot resolve structural features at the molecular level. Time, however, can be exploited to distinguish fluorophores within the same subdiffraction area, if their fluorescence can be switched independently, and reconstruct sequentially their spatial distribution. In this context, photochromic transformations can be invoked to switch fluorescence under optical control. Indeed, fluorescent and photochromic components can be integrated within the same molecular construct or operated within a common supramolecular matrix to produce photoswitchable fluorescent assemblies. In the resulting systems, electronic communication between the fluorescent and photochromic components can be designed to occur in the excited or ground state in order to photodeactivate or photoactivate fluorescence respectively. In the first instance, intercomponent electron or energy transfer processes can be engineered to quench fluorescence after the photochromic transformation. In the second instance, bathochromic shifts can be imposed on the fluorescent component with the photochromic transformation to permit its excitation and activate fluorescence. Both mechanisms can be exploited to overcome diffraction, on the basis of patterned illumination or stochastic localization respectively, and permit the reconstruction of images with resolution down to the nanometer level. Thus, fluorophore-photochrome constructs might well evolve into valuable molecular probes for the investigation of biological samples with nanoscaled resolution.