Intrinsic blinking of red fluorescent proteins for super-resolution microscopy

Klementieva, Natalia V.; Pavlikov, Anton I.; Moiseev, Alexander A.; Bozhanova, Nina G.; Mishina, Natalia M.; Lukyanov, Sergey; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

doi:10.1039/c6cc09200d

Cited by 20 publications

(20 citation statements)

References 23 publications

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“…The partial reversibility of the red form's decay, as observed after the activation, could be attributed to its mixed photobleaching mechanism, which combines both reversible and permanent components. RFPs generally undergo reversible transitions to dark states [52][53][54], which is part of the photobleaching that takes place during fluorescent imaging (these are usually visible at the early phase of the probe photobleaching). Thus, while the reversibility of the chemical red-to-blue conversion after the reductant is washed off could be attributed to the chromophore's re-maturation, the reversibility observed after the dithionite-free "photoconversion" is most likely associated with the red form returning to a bright state.…”

Section: Discussionmentioning

confidence: 99%

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

Protasova

Mishin

Lukyanov

et al. 2021

Photochem Photobiol Sci

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mKate red-to-green photoconversion is a non-canonical type of phototransformation in fluorescent proteins, with a poorly understood mechanism. We have hypothesized that the daughter mKate2 protein may also be photoconvertible, and that this phenomenon would be connected with mKate(2) chromophore photoreduction. Indeed, upon the intense irradiation of the protein sample supplemented by sodium dithionite, the accumulation of green as well as blue spectral forms is enhanced. The reaction was shown to be reversible upon the reductant's removal. However, an analysis of the fluorescence microscopy data, absorption spectra, kinetics and time-resolved fluorescence spectroscopy revealed that the short-wavelength spectral forms of mKate(2) exist before photoactivation, that their fractions increase light-independently after dithionite addition, and that the conversion is facilitated by the photobleaching of the red chromophore form.

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Section: Discussionmentioning

confidence: 99%

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

Protasova

Mishin

Lukyanov

et al. 2021

Photochem Photobiol Sci

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“…53 Since such conformational switches are often energetically controlled, the excitation dependence of kGSR may originate from the absorption of the excitation photons by dark state species and/or the rise of local temperature due to high irradiance. 28,52,54 FusionRed and its sibling TagRFP-T exhibit fluorescence intermittency in live-cell imaging using TIRF microscopy with camera acquisition timescales of 50 ms. 55 The study demonstrated the potential to achieve a theoretical spatial resolution beyond the diffraction limit (~25-30 nm) with FusionRed using SMLM methods like BALM and SOFI. 55 In our previous work, we developed FusionRed-MQV, a FusionRed variant with 3-fold higher molecular brightness developed using a combination of lifetime-based microfluidic selection and site-directed mutagenesis.…”

Section: Introductionmentioning

confidence: 89%

“…28,52,54 FusionRed and its sibling TagRFP-T exhibit fluorescence intermittency in live-cell imaging using TIRF microscopy with camera acquisition timescales of 50 ms. 55 The study demonstrated the potential to achieve a theoretical spatial resolution beyond the diffraction limit (~25-30 nm) with FusionRed using SMLM methods like BALM and SOFI. 55 In our previous work, we developed FusionRed-MQV, a FusionRed variant with 3-fold higher molecular brightness developed using a combination of lifetime-based microfluidic selection and site-directed mutagenesis. 53 Additionally, we found that the substitution C159V in FusionRed resulted in a brighter variant which showed a monoexponential photobleaching trace contrary to the biexponential behavior exhibited by the parental RFP FusionRed.…”

Section: Introductionmentioning

confidence: 89%

Characterizing Dark State Kinetics and Single Molecule Fluorescence of FusionRed and FusionRed-MQ at Low Irradiances

Mukherjee

Thomas

Wilson

et al. 2022

Preprint

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The presence of dark states causes fluorescence intermittency of single molecules due to transitions between “on” and “off” states. Genetically encodable markers such as fluorescent proteins (FPs) exhibit dark states that make several super-resolved single-molecule localization microscopy (SMLM) methods possible. However, studies quantifying the timescales and nature of dark state behavior for commonly used FPs under conditions typical of widefield and total internal reflection fluorescence (TIRF) microscopy remain scarce and pre-date many new SMLM techniques. FusionRed is a relatively bright red FP exhibiting fluorescence intermittency and has thus been identified as a potential candidate for SMLM. We herein characterize the rates for dark-state conversion and the subsequent ground-state recovery of FusionRed and its 2.5-fold brighter descendent FusionRed L175M M42Q (FusionRed-MQ) at low irradiances (1-10 W/cm2), which were previously unexplored experimental conditions. We characterized the kinetics of dark state transitions in these two FPs by using single molecule blinking and ensemble photobleaching experiments bridged with a dark state kinetic model. We find that at low irradiances, the recovery process to the ground state is minimally light-driven and FusionRed-MQ has a 1.3-fold higher ground state recovery time indicating a conformationally restricted dark-state chromophore in comparison to FusionRed. Our studies indicate that the brighter FusionRed-MQ exhibits higher tendency in dark state conversion, thus it is potentially a better candidate for SMLM applications than its progenitor FusionRed.

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“…We have previously reported that TagRFP and several closely related red fluorescent proteins exhibit fast reversible photoconversion between dark and bright states, which could be utilized for super-resolution imaging [8]. We, therefore, sought to assess the photoconversion in a closely related fluorescent proteinTagBFP [4], which is a blue-emitting variant of TagRFP.…”

Section: Biophotonics In Cancer Researchmentioning

confidence: 99%

A Surprising Photoactivity of Blue Fluorescent Protein TagBFP Allows for Super-Resolution Microscopy

Klementieva¹,

Lukyanov²,

Gorbachev³

et al. 2018

Sovrem Tehnol Med

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The aim of this research was to study photobehavior of a popular blue fluorescent protein TagBFP and apply this marker for superresolution microscopy.Materials and Methods. Photoactivation of TagBFP was examined both in protein solution in vitro and in living cells. Subdiffraction imaging was performed using total internal reflection fluorescence microscopy followed by super-resolution radial fluctuations or singlemolecule localization analysis.Results. We show that TagBFP exhibits blinking behavior upon 405 nm light illumination. Moreover, photoactivation to red-emitting state is occurring in the conditions typically used for TagBFP imaging. The red (photoactivated) form of TagBFP possesses spectral properties similar to TagRFP -a close homologue of TagBFP. We show that both blinking and photoactivation of TagBFP can be utilized for super-resolution imaging. We conclude that photoactivation of TagBFP to red-emitting form should be taken into account in the design of multi-channel imaging experiments involving high-power or prolonged UV illumination.Key words: TagBFP; photoactivation; blue-to-red photoconversion; blinking; super-resolution microscopy.

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Intrinsic blinking of red fluorescent proteins for super-resolution microscopy

Cited by 20 publications

References 23 publications

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

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

Characterizing Dark State Kinetics and Single Molecule Fluorescence of FusionRed and FusionRed-MQ at Low Irradiances

A Surprising Photoactivity of Blue Fluorescent Protein TagBFP Allows for Super-Resolution Microscopy

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