Extremely Bright, Near-IR Emitting Spontaneously Blinking Fluorophores Enable Ratiometric Multicolor Nanoscopy in Live Cells

Abstract

New bright, photostable, emission-orthogonal fluorophores that blink without toxic additives are needed to enable multicolor, live-cell, single-molecule localization microscopy (SMLM). Here we report the design, synthesis, and biological evaluation of Yale 676sb , a photostable, near-IR-emitting fluorophore that achieves these goals in the context of an exceptional quantum yield (0.59). When used alongside HMSiR, Yale 676sb enables simultaneous, live-cell, two-color SMLM of two intracellular organelles (ER + mitochondria) with only a single laser and no chemical additives.

Long-term, super-resolution HIDE imaging of the inner mitochondrial membrane in live cells with a cell-permeant lipid probe

“…High-density, environmentally sensitive (HIDE) probes are small molecules that selectively label organelle membranes and resist photobleaching, leading to exceptionally long time-lapse images of organelles in live cells, even at super-resolution [14][15][16][17][18][19][20][21] . Each HIDE probe consists of two parts (Fig.…”

Silicon functionalization expands the repertoire of Si-rhodamine fluorescent probes

“…1 However, it wasn't until 2008 that this LUMO-lowering effect was shown to apply to long-wavelength xanthene dyes by replacing the bridging oxygen atom with a dimethylsilyl group. 2 Si-rhodamine dyes have been widely studied and optimized ever since, 3–15 and the effect of this modification has recently been shown to extend to other classes of near-IR dye scaffolds. 16,17 Despite this intense interest, and the extension of the bridge-substitution strategy to other elements, 3,18–22 virtually all Si-dyes reported thus far have been confined to dimethylsilyl substitution.…”

Surpassing the Background Barrier for Multidimensional Single-Molecule Localization Super-Resolution Imaging: A Case of Lysosome-Exclusively Turn-on Probe

“…The fluorescence from off-targeted fluorophores, the major source of background, could be canceled by introducing photoinduced electron transfer (PET), , spatial proximate energy transfer donor/accepter, − caging strategies, etc. Rhodamines are on the top constructing lists, as they show an environmentally sensitive spirocyclization balance between bright zwitterion and dark lactone. − Recent remarkable works by Wang and Johnsson , introduced sulfamides to the spirolactam site of rhodamines and deployed their protein-tag ligand derivatives for no-wash super-resolution microscopy. Herten, Wombacher, and co-workers have quenched rhodamines with tetrazine moiety and super-resolution imaged engineered proteins through the biorthogonal strategy.…”