Photoactivatable Fluorescent Dyes with Hydrophilic Caging Groups and Their Use in Multicolor Nanoscopy.

Alexey N Butkevich,Elisa D’Este,Lynn M Ostersehlt,Stefan W Hell,Michael Weber,Angel R Cereceda Delgado

doi:10.1021/jacs.1c09999

Abstract

We propose a series of fluorescent dyes with hydrophilic carbamate caging groups that undergo rapid photoactivation under UV (≤400 nm) irradiation but do not undergo spurious two-photon activation with high-intensity (visible or infrared) light of about twice the wavelength. The caged fluorescent dyes and labels derived therefrom display high water solubility and convert upon photoactivation into validated super-resolution and live-cell-compatible fluorophores. In combination with popular fluorescent markers, multiple (up to six)-color images can be obtained with stimulated emission depletion nanoscopy. Moreover, individual fluorophores can be localized with precision <3 nm (standard deviation) using MINSTED and MINFLUX techniques.

Highlights

We propose a series of fluorescent dyes with hydrophilic carbamate caging groups that undergo rapid photoactivation under UV (≤400 nm) irradiation but do not undergo spurious two-photon activation with high-intensity light of about twice the wavelength
With demonstrated robustness of the hydrophilic caging groups against two-photon activation, we explored the possibility of using HCage dyes for multiplexing experiments (Figure 2, Figure S5)
HCage dye-based probes represent a new tool for multiplexed imaging as they allow for standard sample preparation procedures, are compatible with commercial stimulated emission depletion (STED) microscopes, and do not require data post-processing such as spectral unmixing.14a

Summary

Introduction

We propose a series of fluorescent dyes with hydrophilic carbamate caging groups that undergo rapid photoactivation under UV (≤400 nm) irradiation but do not undergo spurious two-photon activation with high-intensity (visible or infrared) light of about twice the wavelength. Gaining detailed information on the context and synaptic status is especially relevant in the studies of neuronal plasticity.[14] HCage dye-based probes represent a new tool for multiplexed imaging as they allow for standard sample preparation procedures, are compatible with commercial STED microscopes, and do not require data post-processing such as spectral unmixing.14a

Results

Conclusion