Remember frFAST and tfPoppy, the FAST variant and its cognate fluorogenic ligand for protein far-red labelling? You should check this preprint in chemRxiv 2025 by Prof. Péter Kele and team of HUN REN in Budapest. They disclose frFAST and tfPoppy (HPAR-3OM therein) for far-red live-cell STED super-resolution microscopy (SRM).
frFAST and tfPoppy have been developed to meet the high demand for red fluorescent proteins, and furthermore far-red. Indeed the growing use of optical biosensors and optogenetic tools obstruct the spectral window available for biological imaging. Moreover, far-red fluorescent proteins allow imaging deeper in tissues. Indeed, autofluorescence, light scattering and absorbance by endogenous molecules, light toxicicty is reduced in the far-red region.
In this article, the authors report frFAST and HPAR-3OM (a.k.a. tfPoppy) to achieve background-free SRM images in fixed and live mammalian cells. They imaged filopodia in living neuroblastoma cells. Moreover, we successfully imaged mitochondrial outer membrane, microtubules, cytoskeleton and histone proteins in live cell STED microscopy. It is envisioned that the excellent photostability achieved by the presented far-red emitting FAST labelling techniques provides a new alternative for stochastic and deterministic SRM imaging of living cells, even for longer imaging sessions.
frFAST for live-cell STED super-resolution microscopy
Want to give it a try? The Twinkle Factory sells the fluorogen HPAR-3OM under the tradename tfPoppy, reference 570655-250. And plasmids are available from Addgene.
More reading on frFAST and tfPoppy
chemRxiv 2025 – Toward Far-red Emitting Chemogenetic Labelling for Live Cell Super-Resolution Microscopy using Fluorescence-Activating and Absorption-Shifting Tag
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