Biology subjects

Bossi, M. L.

Publications and source records attributed to Bossi, M. L..

3 recordsLinked to original sources

bioRxiv2022

Photoactivatable large Stokes shift fluorophores for multicolor nanoscopy

We designed caging-group-free photoactivatable live-cell permeant dyes with red fluorescence emission and [~]100 nm Stokes shifts based on a 1-vinyl-10-silaxanthone imine core structure. The proposed fluorophores undergo byproduct-free one- and two-photon activation, are suitable for multicolor fluorescence microscopy in fixed and living cells and are compatible with super-resolution techniques such as STED (stimulated emission depletion) and PALM (photoactivated localization microscopy). Use of photoactivatable labels for strain-promoted azide-alkyne cycloaddition and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of imaging channel with another large Stokes shift dye have been demonstrated.

biochemistry↗

bioRxiv2022

Spontaneously blinking fluorophores for accelerated MINFLUX nanoscopy

Spontaneously blinking fluorophores, a class of molecules switching rapidly between a dark and a brightly emitting state, have emerged as a popular core to build fluorescent markers for super-resolution microscopy. With typical on-times in the order of tens of milliseconds, they are most suitable for STORM and related nanoscopy methods. Recent MINFLUX nanoscopy, however, can localize molecules even within a millisecond and achieve an up to ten times higher localization precision. Here, we present a series of spontaneous blinkers with short on-times (1-3 ms) matching MINFLUX recording time-scales. Our design builds upon a silicon rhodamine fluorescent core with a modified thiophene- or a benzothiophene-fused spirolactam fragment, which shifts the spirocyclization equilibrium toward the dark closed form at physiological conditions, imparting cell permeability. Concurrently, we obtain a highly photostable, short-lived open form with bright red emission. Characterizing the blinking behavior of single fluorophores bound to three different protein tags (antibodies, nanobodies, and HaloTag self-labeling enzyme) allowed us to select the best candidate for MINFLUX microscopy. The short on-times speed up MINFLUX localization by up to 30-fold.

biophysics↗

bioRxiv2021

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

The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every superresolution fluorescence microscopy (nanoscopy) technique, and the exploration of radically new switching mechanisms remains critical to boosting the performance of established, as well as emerging superresolution methods. Photoactivatable dyes offer significant improvements to many of these techniques, but often rely on photolabile protecting groups that limit their applications. Here we describe a general method to transform 3,6-diaminoxanthones into caging-group free photoactivatable fluorophores. These photoactivatable xanthones (PaX) assemble rapidly and cleanly into highly fluorescent, photo- and chemically stable pyronine dyes upon irradiation with light. The strategy is extendable to carbon- and silicon-bridged xanthone analogs, yielding a new family of photoactivatable labels spanning much of the visible spectrum. Our results demonstrate the versatility and utility of PaX dyes in fixed and live-cell fluorescence microscopy, and both coordinate-targeted stimulated emission depletion (STED) and coordinate-stochastic single-molecule localization superresolution microscopy (SMLM).

biochemistry↗