A general strategy to develop cell permeable and fluorogenic probes for multi-colour nanoscopy

Abstract

Live-cell fluorescence nanoscopy is a powerful tool to study cellular biology on a molecular scale, yet its use is held back by the paucity of suitable fluorescent probes. Fluorescent probes based on regular fluorophores usually suffer from low cell permeability and unspecific background signal. We report a general strategy to transform regular fluorophores into fluorogenic probes with excellent cell permeability and low unspecific background signal. The strategy is based on the conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide. This conversion does not affect the spectroscopic properties of the fluorophore but permits it to exist in a dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell permeable spirolactam. Probes based on such fluorophores generally are fluorogenic as the equilibrium shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1000-fold. Using this simple design principle we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy. The work establishes a general strategy to develop fluorogenic probes for live-cell bioimaging.

Enhancing biocompatibility of rhodamine fluorescent probes by a neighbouring group effect

“…The unprecedented permeability of new tubulin probes allowed imaging at nanomolar - picomolar concentrations, which is in sharp contrast to micromolar-high concentrations reported in other studies 11, 12, 13, 35, 36, 37, 38 . At such low concentrations, no washing step was required even with probes of low fluorogenicity, such as 4-TMR-LTX which can be used as a selective stain for the centrosome at picomolar range.…”

“…Several studies have used dye D 50 to guide rational fluorophore design. 11, 12, 13, 35 . In our case this would not have worked; with the exception of 4-SiR-COOH , dye D 50 values of 4’-, 5’- and 6’-isomers are very close to each other, thus implying no major improvement at the free fluorophore level.…”

“…Regular rhodamines and carbon-substituted analogues (carbopyronines) have the equilibrium shifted towards zwitterion form, which results in relatively poor membrane permeability 8, 9 . To overcome this issue, several studies attempted to induce spirolactone form preference by introducing electron-withdrawing groups into the xanthene core 10, 11, 12 or in the benzoic acid substituent 13 . However, these approaches result in a bulkier core structure and alter the physicochemical properties of the dyes.…”