Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of longer tracks in sptPALM with minimum effort.
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Crystal structures of mEos4b in its green, red and long-lived dark state are available in the Protein Data Bank with accession codes 6GOY, 6GP0 and 6GP1, respectively. The main figures of the paper have associated raw data. Raw data for Supplementary Figures are available upon request. All unique biological materials used in this study and the data that support the findings are available from the corresponding authors upon reasonable request.
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This work was supported by the Agence Nationale de la Recherche (grant no. ANR-17-CE11-0047-01 to D.B.) and used the M4D imaging platform of the Grenoble Instruct-ERIC Center (ISBG: UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (grant no. ANR-10-INBS-05-02) and GRAL (grant no. ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). E.D.Z. thanks the Research Foundation Flanders (FWO) for a doctoral fellowship and travel bursary. S.H. thanks the FWO for a postdoctoral fellowship. We acknowledge support from the European Research Council for ERC Starting Grant NanoCellActivity and the FWO for grant G0B8817N (both to P.D.). The authors thank the staff of beamline ID-23-1 from the ESRF, Grenoble, France. We acknowledge W. Vandenberg, I. Arnal and U. Endesfelder for insightful discussions. We thank N. Zala, J.P. Kleman, A. Conte-Daban and I. Govaerts for practical assistance. Part of the SPT analysis was conducted using resources provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation Flanders (FWO) and the Flemish Government, Department for Economy, Science and Innovation (EWI).
The authors declare no competing interests.
Peer review information: Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Supplementary Figs. 1–30, Supplementary Tables 1–4 and Supplementary Notes 1–9
Supplementary Video 1: MAP4-mEos4b single-particle tracking experiments in the absence of additional 488-nm light.
Clip of the raw data from a sptPALM experiment. The sptPALM experiments with mEos4b were performed under 561-nm (55 W cm–2, 40 ms) and 405-nm (about 0.8 mW cm–2) illumination. (Movie plays at half speed: 12.5 frames per second, field of view: 24 ×24 µm2). The movie is representative of n > 10 cells.
Supplementary Video 2: MAP4-mEos4b single-particle tracking experiments using additional 488-nm illumination.
Clip of the raw data from a sptPALM experiment. The sptPALM experiments with mEos4b were performed under 561-nm (55 W cm–2, 40 ms) with additional 488-nm illumination (4.80 W cm–2). (Movie plays at half speed: 12.5 frames per second, field of view: 24 × 24 µm2). The movie is representative of n > 10 cells.
Clip of the raw data from a sptPALM experiment. The sptPALM experiment with HaloTag–MAP4 labeled with the HaloTag ligand PA-JF549 was performed under 561-nm (55 W cm–2, 40 ms) and 405-nm (about 1 mW cm–2) illumination. (Movie plays at half speed: 12.5 frames per second, field of view: 24 × 24 µm2). The movie is representative of n > 5 cells.
Supplementary Video 4: Trace of a long track of a MAP4-mEos4b molecule in a single-particle tracking experiments using additional 488-nm illumination.
Clip of the raw data from a sptPALM experiment showing one of the long tracks presented in Fig. Figure 2, and being progressively traced along the frames. (Movie: 12.5 frames s–1; field of view: 4 × 4 µm2). The movie is representative of n >> 100 tracks.
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De Zitter, E., Thédié, D., Mönkemöller, V. et al. Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM. Nat Methods 16, 707–710 (2019). https://doi.org/10.1038/s41592-019-0462-3
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