A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

Abstract

The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon-rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super‐Resolution STED Microscopy in Living Cells

“…The magnitude of the response parallels the increase in fluorescence intensity of the tagged dyes 1 a – k ‐Halo, bound nonspecifically and reversibly to bovine serum albumin, upon addition of the anionic surfactant sodium dodecyl sulfate (SDS, see Figure S4), as shown previously for SiR 6h,6k. Interestingly, when the cationic surfactant cetyltrimethylammonium bromide was added instead of SDS, the fluorescence intensity decreased (Figure S4), suggesting that the fluorogenic response of the dyes 1 g , 1 h , and SiR may (at least partially) be due to interactions of the positively charged tricyclic fluorophore core with the local negative charges on the HaloTag protein surface in immediate proximity to its active site.…”

“…6h As expected, upon increasing the water content, the colorless spirolactone forms of all dyes underwent ring opening to the colored and fluorescent zwitterionic forms. Rhodamine dyes 1 b – d favor the zwitterionic form in all but the most non‐polar solvent systems, whereas the carbopyronines 1 e – h and SiR 1 i exist predominantly in the lactone form in solutions with >50 % dioxane content ( D <34.3).…”

The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

“…The tag binds covalently to O 6 -benzylguanine (BG), resulting in the irreversible transfer of the substituted benzyl group to the reactive thiol within the SNAP tag. BG derivatives bearing fluorophores, biotin, or other groups can be used to label a SNAP-tagged protein (Farr et al, 2009;Maurel et al, 2010;Sun et al, 2011;Lukinavičius et al, 2013).…”

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

“…Condensation of 3 with formaldehyde gave bis(tetrahydroquinolinyl)methane 4 . Lithiation and reaction with (CH 3 ) 2 SiCl 2 installed the desired silicon atom, and subsequent oxidation with KMnO 4 gave Si‐anthrone 5 11a, 12. Previously reported strategies to install the o ‐carboxyphenyl ring were met with limited success (Scheme S1 in the Supporting Information).…”