Correlative dual-alternating-color photoswitching fluorescence imaging and AFM enable ultrastructural analyses of complex structures with nanoscale resolution.

There is a practical motivation for correlating different types of microscopy for revealing complementary information of ultrastructures with resolution beyond the diffraction limit. The correlative microscopy strategy based on the combination of super-resolution fluorescence imaging with atomic force microscopy (AFM) is expected to provide both the specificity and three-dimensional structural information of nanomaterials. Herein we synthesized a dual-alternating-color photoswitchable fluorescent probe based on a naphthalimide-spiropyran dyad (NI-SP) and explored the capability of such correlative microscopy for visualizing nanostructures with complex structural hierarchy. NI-SP underwent reversible photoswitching between green and red fluorescence based on a reversible photochemical reaction and such reaction-linked correlation between two distinct types of fluorescence signals intrinsically enabled mutual authentication in super-resolution fluorescence imaging. Additionally, such correlative microscopy also demonstrated mutual complementation between different pieces of structural information of the target acquired via fluorescence imaging and AFM, respectively, in which the former reveals spatial distribution of fluorescent dyes in the nanoscale polymer fibroid micelles while the latter maps the topographical structure of the target with complex structural hierarchy. The results obtained in this work proclaimed that the combination of such correlative microscopy with our NI-SP probe is an effective modality for ultrastructural analysis and has future applications in various complex systems such as tissue/organ imaging.