Research ArticleRESEARCH METHODS

AFM-STED correlative nanoscopy reveals a dark side in fluorescence microscopy imaging

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Science Advances  19 Jun 2019:
Vol. 5, no. 6, eaav8062
DOI: 10.1126/sciadv.aav8062
  • Fig. 1 Correlative imaging.

    (A) Images of the same area are acquired in different modes. The confocal and STED images are acquired in reflection, while the AFM probe reaches the sample from above, providing a three-dimensional topographical view at high resolution. (B) Amyloid fibrils from bovine insulin labeled with fluorescence dye ATTO 488 NHS ester with a 1:19 dye-to-protein ratio. The STED and confocal images are overlaid with AFM topography. The resolution of the STED microscopy image is significantly enhanced with respect to the confocal microscopy image. At the same time, the AFM provides the topographical image on the same sample area. Some fibrillar aggregates are not displayed in fluorescence microscopy (e.g., white arrow). Scale bar, 1 μm.

  • Fig. 2 Insulin fibrils at a dye-to-protein ratio of 1:19.

    Insulin fibril (dye-to-protein ratio, 1:19) images with AFM (A), STED microscopy (B), and the correlative images obtained by (A) and (B) (C). Only a fraction of the fibrillary aggregates is displayed in the optical image. In particular, some fibrils are completely missing, indicating that the labeled monomers were not taking part in the formation of these aggregates. On the contrary, a continuous distribution of fluorophores is present in other aggregates. The ratio of colocalization for this particular field of view is indicated by the histogram. Scale bar, 5 μm. Inset (C), overlay graph.

  • Fig. 3 Insulin fibrils at dye-to-protein ratios of 1:99 and 1:499.

    Insulin fibrils obtained from 1:99 (A to C) and 1:499 (D to F) dye-to-protein ratios. AFM images (A and D), STED microscopy images (B and E), and correlative images (C and F). Several fibrils are totally unlabeled, while a homogeneous fluorescence is emitted by some other aggregates. The ratio of colocalization is shown by the histograms (C and F, insets). Scale bars, 2 μm.

  • Fig. 4 Correlative images of fibrils from Aβ.

    Amyloid aggregates from Aβ1–42 (A to I). Different concentrations of fluorescent peptides were present. The dye-to-protein ratios were 1:19 (A to C), 1:99 (D to F), and 1:499 (G to I). In addition, at the higher dye-to-protein ratio (A to C), some fibrils were not displayed by fluorescence images (e.g., see the white arrows). For amyloid aggregates from Aβ1–40, the dye-to-protein ratio is 1:19 (J to L). Scale bars, 2 μm.

  • Fig. 5 Correlative images from immunolabeled samples.

    (A to F) Amyloid aggregates formed from Aβ1–42 in the absence of fluorescent monomers and subsequently labeled via indirect immunolabeling. All the features displayed in the AFM images are also visible in the STED images and are colocalized. The fibril size, measured by AFM, is larger with respect to the unlabeled fibrils, confirming the presence of a large amount of antibody on the fibrillar aggregates. Scale bars, 2 μm (A to C) and 1 μm (D to F).

  • Fig. 6 Summary and ratios of colocalization.

    (A) A stochastic aggregation of the labeled/unlabeled peptides should bring to the formation of uniformly labeled fibrils. Our experiments indicate a different scenario where only a fraction of the fibrils has a labeled component. Other fibrils, dark in the STED analysis, are unlabeled. These two products are the results of different aggregation pathways. (B) Table showing the ratio of colocalization calculated for the different peptide species and dye-to-protein ratios.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/6/eaav8062/DC1

    Fig. S1. AFM and ThT study of insulin aggregation process.

    Fig. S2. Correlative images of insulin fibrils on a smaller area.

    Fig. S3. Colocalization analysis between AFM and STED images.

    Fig. S4. Confocal microscopy images.

    Fig. S5. Aβ after resuspension of the aggregation medium.

    Fig. S6. Amyloid aggregates from Aβ peptides.

    Fig. S7. Example of correlation between AFM-confocal and AFM-STED images.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. AFM and ThT study of insulin aggregation process.
    • Fig. S2. Correlative images of insulin fibrils on a smaller area.
    • Fig. S3. Colocalization analysis between AFM and STED images.
    • Fig. S4. Confocal microscopy images.
    • Fig. S5. Aβ after resuspension of the aggregation medium.
    • Fig. S6. Amyloid aggregates from Aβ peptides.
    • Fig. S7. Example of correlation between AFM-confocal and AFM-STED images.

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