A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)‐Enhanced Photostability and a Narrow Band of Emission

Abstract

We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.

5-Arylvinylene-2,2′-bipyridyls: Bright “push–pull” dyes as components in fluorescent indicators for zinc ions

“…17), the FRET donor AVB component (blue) is sensitive to zinc(II) coordination [125]. However, it suffers from low photostability, due in part to its propensity for photo-isomerization.…”

“…Moreover, the extent of trans-cis photoisomerization is significantly reduced, which can be attributed to a fast FRET that competes with donor isomerization in the excited state (Fig. 17b) [125]. With a morpholino group that targets lysosomes in mammalian cells, the emission of 41 is shown to correlate with the zinc(II) increase in lysosomes using confocal fluorescence microscopy (Fig.…”

“…(b) Jablonski diagram to show how FRET (from D to A) protects a donor fluorophore from photobleaching. (c) Green FRET dye 41 [125]; and (d) red dye 42 [126]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.…”

“…Panels (a) and (b) are reproduced from Ref. [125] with permission from John Wiley and Sons; panels (c) and (d) are reproduced from Ref. [126] with permission from The Royal Society of Chemistry.…”

“…Reproduced from Ref. [125] with permission from John Wiley and Sons. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.…”