# Bathochromic Shift via C=O to C=S Substitution: A Far-Red Fluorogen for Multiplexed FLIM with FAST Fluorogen-Activating Protein

## TL;DR

Scientists developed a new far-red fluorogen that allows for detailed imaging of multiple proteins in living cells using advanced microscopy techniques.

## Contribution

A novel fluorogen with a bathochromic shift was created, enabling multiplexed FLIM with FAST fluorogen-activating protein.

## Key findings

- HBTR-3,5-DOM exhibits absorption and emission maxima at 640–650 nm and ~670 nm when bound to FAST variants.
- The fluorogen enables genetically encoded live-cell imaging of various subcellular structures.
- Multiplexed imaging of two targeted proteins was demonstrated using FLIM.

## Abstract

The palette of the fluorogen-activating protein FAST expanded into the far-red region by the development of a novel fluorogen, HBTR-3,5-DOM. This was achieved through a C=O to C=S substitution in the classic hydroxybenzylidene-rhodanine core, which induced a bathochromic shift of over 100 nm. The complexes of HBTR-3,5-DOM with FAST variants pFAST and F62L are characterized by absorption and emission maxima at 640–650 nm and ~670 nm, respectively, and are found to exhibit distinct fluorescence lifetimes. The fluorogen is successfully applied in genetically encoded live-cell imaging together with these FAST variants for various subcellular structures. Furthermore, its potential for multiplexed imaging is demonstrated by the simultaneous discrimination of two targeted proteins using fluorescence lifetime imaging microscopy (FLIM).