Novel fluorophores for advancing super-resolution and correlative microscopy

Optical and electronmicroscopy are fundamental imaging techniques in life and material sciences.However, conventional optical microscopy is constrained by the diffractionlimit (~200 nm), preventing visualization of fine subcellular structures.Single-molecule localization microscopy (SMLM) overcomes this limitation byexploiting photoswitchable fluorophores to achieve nanometre-scale resolution.Further expanding SMLM applications and enabling super-resolution correlativelight and electron microscopy (SR-CLEM) require intrinsically blinking andchemically stable fluorophores.
This thesis reportsthe development of two classes of such fluorophores—nanographenes and rylenes—anddemonstrates their utility in advanced imaging. Hydrophilic and functionalizednanographenes were synthesized and exhibit environment-independent blinkingunder diverse conditions, enabling SMLM imaging of amyloid fibrils in air,lysosomal dynamics in live cells, and nascent peptide translation in neurons.These studies revealed heterogeneous local translation along axonal branches.
To extend SMLM intothe near-infrared (NIR) region, intrinsically blinking rylene derivatives weredeveloped, offering reduced phototoxicity for live-cell SMLM imaging. Theirexceptional chemical stability allowed successful application in SR-CLEM,retaining robust blinking behavior after osmium tetroxide fixation and Epon resinembedding. Both single- and dual-color SR-CLEM were achieved, and theOsO₄-induced quenching mechanism, involving energy/electron transfer and aninner filter effect, was elucidated, providing valuable guidance for the futuredesign of SR-CLEM-compatible fluorophores.
Overall, this researchestablishes nanographenes and rylenes as robust, intrinsically blinkingfluorophores for SMLM and SR-CLEM. The mechanistic insights and moleculardesigns presented here advance the development of fluorophores and expand thefrontiers of super-resolution microscopy in the life sciences.