A two-step route to strongly absorbing and efficiently orange to deep red fluorescent, doubly B/N-doped, ladder-type pyrrolo[3,2- b ]pyrroles has been developed. We synthesize and study a series of derivatives of these four-coordinate boron-containing, nominally quadrupolar materials, which mostly exhibit one-photon absorption in the 500–600 nm range with the peak molar extinction coefficients reaching 150 000, and emission in the 520–670 nm range with the fluorescence quantum yields reaching 0.90. Within the family of these ultrastable dyes even small structural changes lead to significant variations of the photophysical properties, in some cases attributed to reversal of energy ordering of alternate-parity excited electronic states. Effective preservation of ground-state inversion symmetry was evidenced by very weak two-photon absorption (2PA) at excitation wavelengths corresponding to the lowest-energy, strongly one-photon allowed purely electronic transition. π-Expanded derivatives and those possessing electron-donating groups showed the most red-shifted absorption- and emission spectra, while displaying remarkably high peak 2PA cross-section ( σ 2PA ) values reaching ∼2400 GM at around 760 nm, corresponding to a two-photon allowed higher-energy excited state. At the same time, derivatives lacking π-expansion were found to have a relatively weak 2PA peak centered at ca. 800–900 nm with the maximum σ 2PA ∼50–250 GM. Ourmore »
Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor
Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor’s functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R0) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.
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- International Journal of Molecular Sciences
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- National Science Foundation
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