Abstract
Fluorescence microscopy is an essential tool for understanding dynamic processes in living cells and organisms. However, many fluorescent probes for labelling cellular structures suffer from unspecific interactions and low cell permeability. Herein, we demonstrate that the neighbouring group effect which results from positioning an amide group next to a carboxyl group in the benzene ring of rhodamines dramatically increases cell permeability of the rhodamine-based probes through stabilizing a fluorophore in a hydrophobic spirolactone state. Based on this principle, we create probes targeting tubulin, actin and DNA. Their superb staining intensity, tuned toxicity and specificity allows long-term 3D confocal and STED nanoscopy with sub-30 nm resolution. Due to their unrestricted cell permeability and efficient accumulation on the target, the new probes produce high contrast images at low nanomolar concentrations. Superior performance is exemplified by resolving the real microtubule diameter of 23 nm and selective staining of the centrosome inside living cells for the first time.
Highlights
Modern super-resolution uorescence microscopy and nanoscopy techniques permit observation of biological processes in living organisms down to the molecular level.[1]
We propose a way for modulating the spirolactone–zwitterion equilibrium without any change in the uorescent dye core structure by exploiting the neighbouring group effect (NGE), i.e. the phenomenon that two neighbouring carboxylic groups can in uence each other via steric, electrostatic or H-bond interactions.[7]
The existence of a novel isomeric class of 40carboxyrhodamines was debated due to the synthesis challenge arising from steric hindrance and the ortho-effect altered reactivity of adjacent carbonyl groups.[9]
Summary
We introduce a new positional isomer with NGE to increase the cell permeability and uorogenicity of regular rhodamines without changing their spectroscopic properties. We demonstrate that excellent quality of wash-free multi-colour live-cell images can be obtained at low working concentrations with wide- eld, confocal and super-resolution microscopes even with moderately uorogenic probes. Selective centrosome staining is demonstrated using exceptionally well performing tubulin probes. Superior performance is exempli ed by resolving the real microtubule diameter of 23 nm in STED nanoscopy images
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