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Multitarget super-resolution microscopy with high-density labeling by exchangeable probes

Nature Methods volume 12, pages 743–746 (2015)Cite this article

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An Erratum to this article was published on 28 August 2015

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Abstract

We have developed a multitarget super-resolution microscopy technique called image reconstruction by integrating exchangeable single-molecule localization (IRIS). IRIS uses protein fragment–based probes that directly associate with and dissociate from their targets over durations on the order of tens of milliseconds. By integrating single-molecule localization and sequential labeling, IRIS enables unprecedented labeling density along multiple cellular structures. IRIS can be used to discern the area-specific proximity between cytoskeletal components and focal adhesions within a single cell.

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Figure 1: Super-resolution imaging using IRIS.
Figure 2: Super-resolution images of three cytoskeletons and focal adhesions.
Figure 3: Area-specific proximity between cytoskeletons and focal adhesions.

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  • 03 August 2015

    In the PDF version of this article initially published, the Acknowledgments section erroneously listed the Uehara Memorial Foundation as a funder twice. The error has been corrected in the PDF version of this article.

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Acknowledgements

We thank H. Mizuno and S. Yamashiro (Kyoto University) for rabbit muscle actin and Y. Mimori-Kiyosue (RIKEN) for providing cDNAs encoding EB1, CLIP-170, CLASP2γ and APC. This work was supported by the Cabinet Office of the Government of Japan through the Funding Program for Next Generation World-Leading Researchers (LS013), by the Uehara Memorial Foundation (N.W.) and by Japan Society for the Promotion of Science (JSPS) KAKENHI grant numbers 26440091 and 15H01635 (T.K.).

Author information

Author notes

  1. Masahiro Maruoka

    Present address: Present address: Division of Pathogenetic Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan.,

Authors and Affiliations

  1. Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan

    Tai Kiuchi, Makio Higuchi & Naoki Watanabe

  2. Laboratory of Single-Molecule Cell Biology, Tohoku University Graduate School of Life Sciences, Sendai, Japan

    Akihiro Takamura & Masahiro Maruoka

  3. Laboratory of Single-Molecule Cell Biology, Kyoto University Graduate School of Biostudies, Kyoto, Japan

    Naoki Watanabe

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  1. Tai Kiuchi
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Contributions

T.K. and N.W. designed experiments and wrote the manuscript. T.K., M.H., A.T. and M.M. conducted experiments and analyzed the data.

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Correspondence to Tai Kiuchi or Naoki Watanabe.

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Integrated supplementary information

Supplementary Figure 1 Characterization of Atto 488–Lifeact.

(a) Speckle lifetime distribution of Atto488-Lifeact in fixed XTC cells. The SiMS images were acquired with a 10 ms exposure time. Speckle lifetime measurement was performed using the Speckle TrackerJ plug-in as described previously27,31. The black line shows the single exponential curve fit of the lifetime distribution data between 20 ms and 110 ms with a half-life of 23 ms. The photobleaching rate of Atto488-Lifeact for 200 ms was negligible. (b) Atto488-Lifeact speckles analysed using DAOSTORM (red circles). The SiMS images of Atto488-Lifeact in a cell were acquired with a 50 ms exposure time. Its central position was determined with nanometer accuracy. A large fraction of speckle localizations changed in the next frame. (c) The super-resolved images of a single actin filament in vitro by TIRF microscopy using Atto488-Lifeact. The left image is the same image in Figure 1b, which was reconstructed from only high brightness speckles (top 12% of all measured speckles). The right image was reconstructed from all measured speckles. The labeling density on the filament is indicated at the top of each panel. (d) The cross-sectional profiles of single actin filaments in vitro (n = 10 filaments) in the image reconstructed from high brightness speckles (black bars) and all speckles (white bars). The total label is normalized to 1. The black and grey lines show the Gaussian fit with a FWHM of 23 nm (bright speckles) and 38 nm (all speckles), respectively. Error bar represents ±S.E.M. (e) Coefficient of variation of the labeling density of Atto488-Lifeact along a single actin filament as a function of the labeling density. The coefficient of variation is calculated as the ratio between the standard deviation and the mean of the signal intensity along the filament in the IRIS images reconstructed from 2×103 to 2×105 frames in Figure 1d. The coefficient of variation gradually decreases with increasing labeling density.

Supplementary Figure 2 Comparison of labeling patterns along microtubules between IRIS and other super-resolution techniques.

(a-e) Super-resolved images of microtubules by IRIS using CLIP-170 fragment (a), by STORM using anti-β tubulin (b, c), by STORM using anti-GFP nanobodies for GFP-tagged tubulin (d) and by Exchange-PAINT using anti-β tubulin (e). The right image represents an enlarged image of the microtubule indicated in the left image (between arrowheads). (f) Line profiles of the label intensity along the microtubules in right panels of a-e. The label intensities were normalized by the mean label intensity along the microtubule. The curve of STORM (c) shows the green RGB value along the microtubule. The IRIS image in a is a part of the Epi-illuminated IRIS image in Figure 2b. The STORM (b,c), nanobody (d) and Exchange-PAINT images (e) are reproduced from ref. 16, AAAS (b); ref. 17, Nature Publishing Group (c); ref. 18, Nature Publishing Group (d); and ref. 7, Nature Publishing Group (e). Note the homogeneous labels along the microtubules by IRIS although the FWHM is 60 nm in the IRIS image and is the smallest at 26.9 nm with nanobody-labeled microtubules18.

Supplementary Figure 3 The dwell time of the selected IRIS probes on their targets.

The dwelling times of the probes (n = 100-112) were plotted using a complementary cumulative distribution function (1-Ndissociation) after correction for photobleaching. Ndissociation is the cumulative relative frequency of dissociated probes. The photobleaching rate was measured using GFP-actin expressing cells after fixation. Its half-life was 8 s under the same laser conditions. The decline of dwelling probes was fitted by a single exponential decay function (red line) to determine half-life values. Represented measurements in Supplementary Table 1 are shown.

Supplementary Figure 4 Labeling patterns of IRIS probes on microtubules.

CLIP-170 fragment (residues 3-309), APC fragment (residues 2536-2843), MAP4 fragment (residues 1-908) and Tau isoform 3 full length labeled microtubules. The pattern was altered in accordance with fixation procedure. (a, b) Time-lapse imaging of EB1-EGFP during fixation. EB1-EGFP was fixed on microtubule tips not by treatment with 3.7% PFA and 0.5% Triton X-100 (a), but by treatment with cold methanol (b). (c) The TIRF-illuminated IRIS images of microtubules using CLIP-170 fragment and Tau in PFA and Triton X-100 treated cells. These probes yielded the IRIS images of microtubule lattices. The merged image shows the coincidence of the visualized microtubule lattices network between the two IRIS images. (d) The TIRF-illuminated IRIS images of microtubules using CLIP-170 fragment and APC fragment in cells pretreated with cold methanol for 5 sec before PFA and Triton X-100 treatment. These probes yielded the IRIS images of microtubules where the microtubule tips were visualized with much higher signals than that in the microtubule lattices. The merged image shows the coincidence of the visualized microtubule tips between the two IRIS images. (e) The TIRF-illuminated IRIS images of microtubules using CLIP-170 fragment, MAP4 fragment and Tau in cold methanol-pretreated cells. The microtubule tips labeled strongly with CLIP-170 fragment were labeled weakly with MAP4 fragment and Tau (lower panels).

Supplementary Figure 5 Labeling patterns of IRIS probes on focal adhesions.

(a-f) Paxillin full length (FL), Src fragment (residues 3-251) and PIPKIγ fragment (residues 641-668) labeled focal adhesions. The patterns were compared with the live-cell image of GFP-Paxillin (a), which was used to visualize focal adhesions. After the image acquisition of GFP-Paxillin, the living cells were fixed and permeabilized (b). The remaining fluorescence of GFP-Paxillin was completely photobleached by a strong excitation laser (c). Then the IRIS imaging was carried out in the order of the Src fragment (d), the Paxillin FL (e) and the PIPKIγ fragment (f). (g) The merged image of the live-cell image of GFP-Paxillin in a with the three super-resolved images in d-f. In the merged images, colours indicate the live-cell imaging GFP-Paxillin (grey), the Src fragment (red), the Paxillin FL (green) and the PIPKIγ fragment (blue). (h) Cross-sectional profiles of the focal adhesion in three IRIS images and the live cell image of GFP-Paxillin. The line on the left panel of g indicates the measured area. Scale bar in a applies to b-g.

Supplementary Figure 6 Super-resolved image of microtubules.

(a) Microtubules in fixed and permeabilized XTC cells were imaged by TIRF microscopy using a CLIP-170 fragment. The probes were excited by the simultaneous illumination of 473 nm and 488 nm laser lines to obtain strong signals from each speckle. In addition, the IRIS image is reconstructed from high brightness speckles (top 12%) to improve the localization accuracy. (b) The cross-sectional profile of microtubules (n = 10, grey bars). The total label is normalized to 1. The black curve shows the Gaussian fit to the mean profile, with a FWHM of 41 nm. This FWHM is wider than the FWHM of a single actin filament (23 nm) obtained by Atto488-Lifeact in this study. This difference is probably attributable to the large size of microtubules (diameter, 25 nm). Error bars represent the S.E.M.

Supplementary Figure 7 z profile of TIRF excitation intensity.

(a) The fluorescence images of HyLight488-labeled microtubules acquired by TIRF and epi illumination microscopy. The epi images were acquired as a z-stack image (0.2 μm step size). The z-stacked epi images were used to determine the z distance of each point along the tilted microtubule (see Methods). Scar bar, 5 μm. (b) Z profile of TIRF excitation intensity. The z profile was characterized by associating the z distance of each point with the ratio of the intensity of the TIRF-illuminated microtubule and that of the epi-illuminated microtubule at the bottom from three labeled microtubules (see Methods).

Supplementary Figure 8 z-position maps of three cytoskeletons.

(a-c) The z position maps of actin bundles (a), microtubules (b) and intermediate filaments (c) were calculated from the TIRF- and epi-illuminated IRIS images in Figure 2a-c (see Methods). The right image represents an enlarged image of the boxed area shown in the left image. In the right image of c, intermediate filaments form mesh-like structures throughout the cell body, and some filaments are located at a height of approximately 200 nm behind the lamellipodium base (white ellipse). (d) The height of actin filaments in lamellipodia (LP), stress fibres (SF) and actin arcs (Arc). These filaments are distributed with a centroid height (mean ± S.D.) of 42 ± 43 nm, 34 ± 30 nm and 217 ± 132 nm, respectively. (e) Line profile of z positions along the microtubule (arrowheads) between “S” and “E” in b. The microtubule submerges toward the cell periphery from 150-200 nm to 50-100 nm.

Supplementary Figure 9 The movement of microtubule tips in the vicinity of actin stress fibers and focal adhesions.

(a) The super-resolved epi image (left) and TIRF images (right) of microtubules in the central area of ROI2 in Figure 2e. In the CLIP-170 fragment-visualized microtubules, the signal is lost in places (asterisks). (b) The z position map of microtubules in a (upper) and its merged images with the super-resolved TIRF image of focal adhesions (lower left) and actin filaments (lower right). The z positions of microtubules are calculated from the signal intensity ratio between epi and TIRF images (see Methods). Scale bar in a applies to b. (c) Line profiles of the z position of the microtubule (magenta diamonds) and the intensity of actin filaments and focal adhesions on a line along the microtubule (yellow arrowheads) between “S” and “E” in b. The magenta curve shows a moving average of four data points for the z position of the microtubule. (d) The z position map of microtubules (left) and its merged image (right) with the super-resolved TIRF images of actin filaments (grey) and focal adhesions (magenta). Focal adhesions are visualized by the combination of the Src fragment (residues 3-251) and the Paxillin full length. (e) The left panels show live-cell epi images (red) and TIRF images (green) of EB1-EGFP acquired prior to IRIS imaging. The middle and right panels show the z position and the local speed of the EB1-labeled microtubule tip, respectively, superimposed on the actin filaments and focal adhesions shown in d. The growth of the microtubule tip slowed down on contact with the stress fibres (arrowheads). The microtubule analysed with the EB1 trajectory is indicated by an asterisk in d.

Supplementary Figure 10 The movement of microtubule tips in the vicinity of actin stress fibers.

(a) The z position map of microtubules (upper) and its merged image (lower) with the super-resolved TIRF images of actin filaments (grey). (b) The left panels show live-cell epi images (red) and TIRF images (green) of EB1-EGFP acquired prior to IRIS imaging. The middle and right panels show the z position and the local speed of the EB1-labeled microtubule tip, respectively, superimposed on the super-resolved images shown in a. The microtubule analysed with the EB1 trajectory is indicated by arrowheads in a and white arrows in b. The growth of the microtubule tip slowed down on contact with the stress fibres (arrowhead) and then changed direction laterally (magenta arrows). Scale bar in a applies to b.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Table 1 and Supplementary Notes 1 and 2 (PDF 16851 kb)

Sequential presentation of the super-resolved images and the merged image.

These images are shown in Figure 2a-e. Scale bar, 10 μm. (MOV 18736 kb)

Live-cell imaging of EB1-EGFP superimposed on the super-resolved image of actin filaments near focal adhesions.

The merged movie of the epi-imaging (red) and TIRF-imaging (green) of EB1-EGFP is shown on the top. Its superimposed movie on the super-resolved image of actin filaments is shown on the bottom. These data are shown in Supplementary Figure 9e. The images were acquired at 1 s intervals. Scale bar, 1 μm. (MOV 6209 kb)

Live-cell imaging of EB1-EGFP superimposed on the super-resolved image of actin filaments.

The merged movie of the epi-imaging (red) and TIRF-imaging (green) of EB1-EGFP is shown on the left. Its superimposed movie on the super-resolved image of actin filaments is shown on the right. These data are shown in Supplementary Figure 10b. The images were acquired at 1 s intervals. The EB1-labeled microtubule tip analysed in Supplementary Figure 10b is indicated by arrowheads. Scale bar, 1 μm. (MOV 9578 kb)

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Kiuchi, T., Higuchi, M., Takamura, A. et al. Multitarget super-resolution microscopy with high-density labeling by exchangeable probes. Nat Methods 12, 743–746 (2015). https://doi.org/10.1038/nmeth.3466

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