Facile autofluorescence suppression enabling tracking of single viruses in live cells

Chen, Yen-Cheng; Sood, Chetan; Francis, Ashwanth C.; Melikyan, Gregory B.; Dickson, Robert M.

doi:10.1074/jbc.ra119.010268

Cited by 6 publications

(6 citation statements)

References 51 publications

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“…Photoswitchable FPs (PS-FPs) can also give background-free detection by modulating both primary and secondary lasers and detecting at the sum and difference of the modulation frequencies, 34 or by using sequential excitation conditions. 19 In each case, demodulating OMFP fluorescence results in background suppression and drastic signal enhancements over autofluorescence and, in some cases, resolution of bound vs unbound OMFPs through kinetic resolution. 34 The palette of OMFPs would benefit from expansion to other colors and modification of the chromophore surroundings to further tune dark state lifetimes.…”

Section: ■ Introductionmentioning

confidence: 99%

“…YFPs, as a spectral class, are among the brightest and most versatile genetically encoded fluorophores, which makes them essential in multilabel/multicolor imaging schemes . As EYFP (10C, but renamed EYFP later) results from only four point mutations (S65G, V68L, S72A, and T203Y) of green fluorescent protein (GFP), − tuning the immediate chromophore environment tailors spectral and photophysical properties for a wide array of imaging applications. − While both electronic and steric , interactions are known to alter emission, longer wavelength emission typically comes from an anionic chromophore, often facilitated through ground or excited state proton transfer pathways, while photoswitching tends to result from cis–trans isomerization. ,, Although genetically optimized for bright emission, fluorescent proteins (FPs) also exhibit multiple dark states that enable new imaging modalities. − The multiple states in EYFP, for example, were demonstrated to be optically interconvertible, even on the single molecule level, laying the groundwork for many advances in super-resolution fluorescence and high-sensitivity imaging. Thus, both proton transfer and isomerization are crucial to FP photophysics and guide design of dark and bright states within FPs, while giving rise to many of their novel applications. , …”

Section: Introductionmentioning

confidence: 99%

“…To date, only blue fluorescent proteins (BFPs) and Aequorea coerulescens GFP (AcGFP) have been shown to be optically modulatable. Photoswitchable FPs (PS-FPs) can also give background-free detection by modulating both primary and secondary lasers and detecting at the sum and difference of the modulation frequencies, or by using sequential excitation conditions . In each case, demodulating OMFP fluorescence results in background suppression and drastic signal enhancements over autofluorescence and, in some cases, resolution of bound vs unbound OMFPs through kinetic resolution .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2021

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“…Reversibly photoswitchable FP (RSFP) can be utilized to suppress the unmodulatable fluorescent background and enhance the signal contrast by means of optical lock-in detection (OLID) [ 19 ], synchronously amplified fluorescence image recovery (SAFIRe) [ 20 , 21 ] and out-of-phase imaging after optical modulation (OPIOM) [ 22 ]. We speculated that similar strategies could be applied with an OsO 4 —and Epon-resistant RSFP to eliminate the resin background and the RFP crosstalk signal.…”

Section: Introductionmentioning

confidence: 99%

Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM

Peng

et al. 2022

Cells

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Post-embedding correlative light and electron microscopy (CLEM) has the advantage of high-precision registration and enables light and electron microscopy imaging of the same slice. However, its broad application has been hampered by the limited available fluorescent proteins (FPs) and a low signal-to-background ratio (SBR). Here, we developed a green photoswitchable FP, mEosEM-E with substantially high on/off contrast in EM samples embedded in Epon resin, which maximally preserves cellular structures but quenches the fluorescence of FPs. Taking advantage of the photoswitching property of mEosEM-E, the autofluorescence background from the resin was significantly reduced by a subtraction-based CLEM (sCLEM) method. Meanwhile, we identified a red fluorescent protein (RFP) mScarlet-H that exhibited higher brightness and SBR in resin than previously reported RFPs. With mEosEM-E and mScarlet-H, dual-colour post-Epon-embedding CLEM images with high SBR and no cross-talk signal were successfully performed to reveal the organization of nucleolar proteins. Moreover, a dissection of the influences of different EM sample preparation steps on the fluorescence preservation for several RFPs provides useful guidance for further probe development.

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“…Reversibly photoswitchable FP (RSFP) can be utilized to suppress the unmodulatable fluorescent background and enhance the signal contrast by means of optical lock-in detection (OLID) [19], synchronously amplified fluorescence image recovery (SAFIRe) [20, 21] and out-of-phase imaging after optical modulation (OPIOM) [22]. We speculated that similar strategies could be applied with an OsO 4 - and Epon-resistant RSFP to eliminate the resin background and the RFP crosstalk signal.…”

Section: Introductionmentioning

confidence: 99%

Improved fluorescent proteins for dual-color post-embedding CLEM

Peng

et al. 2022

Preprint

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Post-embedding correlative light and electron microscopy (CLEM) has the advantage of high-precision registration and enables light and electron microscopy imaging of the same slice. However, its broad application has been hampered by the limited available fluorescent proteins (FPs) and low signal-to-background ratio (SBR). Here, we developed a green photoswitchable FP, mEosEM-E with substantially high on/off contrast in EM samples embedded in Epon resin which maximally preserves cellular structures but quenches the fluorescence of FPs. Taking advantage of the photoswitching property of mEosEM-E, the autofluorescence background from the resin was significantly reduced by a subtraction-based CLEM (sCLEM) method. Meanwhile, we identified a red fluorescent protein (RFP) mScharlet-H that exhibited higher brightness and SBR in resin than previously reported RFPs. With mEosEM-E and mScharlet-H, dual-color post-Epon-embedding CLEM images with high SBR and no cross-talk signal were successfully performed to reveal the organization of nucleolar proteins. Moreover, a dissection of the influences of different EM sample preparation steps on the fluorescence preservation for several RFPs provides useful guidance for further probe development.

show abstract

Facile autofluorescence suppression enabling tracking of single viruses in live cells

Cited by 6 publications

References 51 publications

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

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

Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM

Improved fluorescent proteins for dual-color post-embedding CLEM

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