Fluorescent microscopy became one of the key tools in biology and drug discovery, therefore, the need for brighter and more efficient fluorescent stains is ever growing. While many fluorescent probes have been developed to provide insights into different cellular processes, the used fluorophores remained highly conservative. Although they determine many characteristics of probes, the fluorophores utilized are constrained to only a few examples, such as cyanines, rhodamines, coumarins, BODIPYs and fluorescein derivatives. For example, in the most commonly used green channel of microscopes, the latter two are available and while they offer many advantages, they are compromised by considerable spectral overlap between their absorbed and emitted light. The small Stokes-shift leads to a large part of the emitted light being filtered out to avoid a high background caused by scattering of the excitation source. Maintaining a high brightness and a high Stokes-shift at the same time is however challenging, requiring novel fluorophores instead just fine-tuning existing ones. In this work we have demonstrated, that compounds based on a novel heterocyclic system, chromeno[2,3-d]imidazole scaffold, exhibit bright fluorescent intensity (&gt;3×104) and a reasonable Stokes-shift (&gt;70 nm), that eliminates spectral overlap. The presented structure can be considered a conformationally locked GFP-chromophore, which results in a green (λ = 465 nm) excitation wavelenght. We present a straightforward, one-pot synthesis by utilizing an intramolecular ring closure aided by microwave irradiation. Detailed spectroscopical characterization of the fluorophores are presented and their practical relevance is demonstrated by confocal and two-photon live cell imaging using targeted fluorescent probes built on the chromeno[2,3-d]imidazole system.

Chromeno[2,3-d]imidazoles: A novel heteroaromatic scaffold as a highly efficient fluorophore derived from the chromophore of the GFP: Synthesis, spectroscopy and application in fluorescent microscopy

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