Optically Modulated and Optically Activated Delayed Fluorescent Proteins through Dark State Engineering

Abstract

Modulating fluorescent protein emission holds great potential for increasing readout sensitivity for applications in biological imaging and detection. Here, we identify and engineer optically modulated yellow fluorescent proteins (EYFP, originally 10C, but renamed EYFP later, and mVenus) to yield new emitters with distinct modulation profiles and unique, optically gated, delayed fluorescence. The parent YFPs are individually modulatable through secondary illumination, depopulating a long-lived dark state to dynamically increase fluorescence. A single point mutation introduced near the chromophore in each of these YFPs provides access to a second, even longer-lived modulatable dark state, while a different double mutant renders EYFP unmodulatable. The naturally occurring dark state in the parent YFPs yields strong fluorescence modulation upon long-wavelength-induced dark state depopulation, allowing selective detection at the frequency at which the long wavelength secondary laser is intensity modulated. Distinct from photoswitches, however, this near IR secondary coexcitation repumps the emissive S1 level from the long-lived triplet state, resulting in optically activated delayed fluorescence (OADF). This OADF results from secondary laser-induced, reverse intersystem crossing (RISC), producing additional nanosecond-lived, visible fluorescence that is delayed by many microseconds after the primary excitation has turned off. Mutation of the parent chromophore environment opens an additional modulation pathway that avoids the OADF-producing triplet state, resulting in a second, much longer-lived, modulatable dark state. These Optically Modulated and Optically Activated Delayed Fluorescent Proteins (OMFPs and OADFPs) are thus excellent for background- and reference-free, high sensitivity cellular imaging, but time-gated OADF offers a second modality for true background-free detection. Our combined structural and spectroscopic data not only gives additional mechanistic details for designing optically modulated fluorescent proteins but also provides the opportunity to distinguish similarly emitting OMFPs through OADF and through their unique modulation spectra.

Sequential Two-Photon Delayed Fluorescence Anisotropy for Macromolecular Size Determination

“…34,35 This self-assembled macromolecular assembly forms a highly stable container 34−36 that can be used for cargo delivery, 27,30 and VLPs were loaded with the fluorescent protein mVenus�a fluorescent protein we have shown to exhibit OADF. 21,36,37 Qβ-mVenus OADFA yields a measured radius of 12.5 ± 0.6 nm, which is very close to the radius measured by dynamic light scattering (DLS) (Figure 5C) and TEM (Figure S3).…”

“…For better control of particle size and more biological relevance, we used OADFA to determine the size of Qβ, a recombinant virus-like particle (VLP) derived from an E. coli bacteriophage (Leviviridae) . , This self-assembled macromolecular assembly forms a highly stable container − that can be used for cargo delivery, , and VLPs were loaded with the fluorescent protein mVenusa fluorescent protein we have shown to exhibit OADF. ,, Qβ-mVenus OADFA yields a measured radius of 12.5 ± 0.6 nm, which is very close to the radius measured by dynamic light scattering (DLS) (Figure C) and TEM (Figure S3).…”

“…The mVenus expression followed the procedure published in previous work . N-terminal His 6 -tag mVenus plasmid (gifted by Prof. Fahrni) was transformed into chemically competent E. coli BL21­(DE3) cells via heat shock.…”

“…The mVenus expression followed the procedure published in previous work. 21 N-terminal His 6 -tag mVenus plasmid (gifted by Prof. Fahrni) was transformed into chemically competent E. coli BL21(DE3) cells via heat shock. Single colonies were selected and cells were grown in the presence of kanamycin at 37 °C with shaking at 250 rpm to an optical density at 600 nm (OD 600 ) of 0.6.…”

Characterizing dark state kinetics and single molecule fluorescence of FusionRed and FusionRed-MQ at low irradiances

“…[39][40][41] Dark states can be selectively accessed or depopulated using optical or thermal modulation. [42][43][44][45][46][47][48] For example, pulsed excitation with resonant frequencies was used to populate and depopulate the dark state in FPs like AcGFP and rsFastLime, providing selective modulation of fluorescence. 45 Varied mechanisms of accessing dark states as listed above have made FPs natural choices for several SMLM techniques.…”