Chromophore reduction plus reversible photobleaching: how the mKate2 “photoconversion” works

Protasova, Elena A.; Mishin, Alexander S.; Lukyanov, Konstantin A.; Maksimov, Eugene G.; Bogdanov, Alexey M.

doi:10.1007/s43630-021-00060-8

Cited by 8 publications

(7 citation statements)

References 54 publications

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“…We are happy to announce that today, this virtual volume unites 18 contributions by scientists from 17 countries (Canada, Czech republic, Finland, France, Germany, Hungary, India, Italy, Japan, Lebanon, Poland, Russia, Spain, Switzerland, Taiwan, UK, US) touching upon the wide variety of the subjects dealt with by Klaus at various times [25,[127][128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143].…”

Section: Legacy Tribute Continuation…mentioning

confidence: 99%

“…A paper from Protasova et al investigates the mechanism of colour change in mKate2, an engineered red fluorescent protein used as popular marker in cell biology [137]. The authors found that the unsual red-to-green transformation is in fact due to a pre-existing green fraction that becomes better visible upon bleaching of the red form.…”

Section: Legacy Tribute Continuation…mentioning

confidence: 99%

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Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer

Mathis

Sage

Byrdin

2022

Photochem Photobiol Sci

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Time-resolved absorption spectroscopy is a powerful tool to unravel biological functions and has been a key technology for elucidating the working of electron transfer chains in photosynthesis or photorepair of UV-damaged DNA. Both of these areas have seen important contributions from laboratories all over the world, not the least of them stemming from the ingenious technical advances described by Klaus Brettel, first at the Technical University of Berlin (Germany), and later at the Atomic Energy Agency in Saclay (France). Now, after more than forty years of tireless scientific activity, Klaus is approaching retirement and this collection gathers together tributes in the form of scientific contributions from colleagues along the way, covering a spectrum of topics as diverse as photosynthesis, light-induced DNA repair, electron and proton transfer in light signalling, flavin based photo-enzymology, fluorescent marker photophysics, synthetic models and modelisation, delicate sample transient absorption spectroscopy. In an era where science is increasingly changing context from "fundamental" to "applied", Klaus' curiosity and tenacity worked hand in hand in a most effective manner to further both technical possibilities and basic understanding.

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Section: Legacy Tribute Continuation…mentioning

confidence: 99%

Section: Legacy Tribute Continuation…mentioning

confidence: 99%

Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer

Mathis

Sage

Byrdin

2022

Photochem Photobiol Sci

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“…A further investigation showed that the crosstalk phenomenon is universal for all the tested RFPs that survived standard EM sample preparation (Figure S8). A very likely reason is that under high-energy illumination, severe chromophore photoreduction happens that leads to obvious red-to-green photoconversion of the RPFs, a mechanism that has been reported before [24–26].…”

Section: Resultsmentioning

confidence: 95%

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.

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“…A further investigation showed that the crosstalk phenomenon is universal for all the tested RFPs that survived standard EM sample preparation ( Figure S2 ). A very likely reason is that under high-energy illumination, severe chromophore photoreduction happens that leads to obvious red-to-green photoconversion of the RPFs, a mechanism that has been reported before [ 25 , 26 , 27 ].…”

Section: Resultsmentioning

confidence: 96%

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.

show abstract

Chromophore reduction plus reversible photobleaching: how the mKate2 “photoconversion” works

Cited by 8 publications

References 54 publications

Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer

Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer

Improved fluorescent proteins for dual-color post-embedding CLEM

Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM

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