Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

Abstract

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, which poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell stimulated emission depletion (STED) microscopy, or into a spontaneously blinking dye for single molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

A general design of caging-group-free photoactivatable fluorophores for live-cell nanoscopy

“…Rhodamine dyes have emerged as some of the most widely employed fluorophores in fluorescence microscopy and nanoscopy due to the remarkable tunability of their optical and chemical properties [13][14][15] , cell membrane permeability 16 , photostability 17 and brightness 18 . In particular, silicon rhodamines 19 are often favoured for their intrinsic redshifted emission, fluorogenic behaviour 20 and live-cell compatibility [21][22][23] .…”

Malaria parasite centrins assemble by Ca2+-inducible condensation

“…To address whether centrin might be dynamically reorganized within this diffraction-limited region we implemented live cell STED using a parasite line expressing PfCen1 tagged with Halo (Fig. S1) and labeled with the MaP-SiR-Halo fluorogenic dye ( 22 ) (Fig. 1C-D).…”

How Single-Molecule Localization Microscopy Expanded Our Mechanistic Understanding of RNA Polymerase II Transcription

“…The advantage of rhodamine-based fluorophores is that they exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. This feature of rhodamine dyes and their derivatives can be differently utilized in various SRM modalities that have different requirements of the dynamic equilibrium between the zwitterion and spirocyclic rhodamine forms [54,55].…”

“…This approach is particularly useful for the imaging of individual molecules in the densely packed nucleus. Progressive development of finely-tuned fluorophores [55] including fluorophores with very specific features, such as photosensitizers that generate reactive oxygen species [79] for delicate applications and allowing for multiplexing at a high spatial and temporal resolution [54] continues to push the boundaries of SMLM applications. Moreover, the fluorescent signal of individual molecules of interest can be amplified by multivalent tags such as Sun-tag [80] or repetitive HaloTag [71,78].…”