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Correlative light microscopy for high-content screening
 
Benjamin Flottmann*1, Manuel Gunkel*1, Tautvydas Lisauskas*1, Mike Heilemann1,2, Vytaute Starkuviene1, Jürgen Reymann1, and Holger Erfle1
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With this in hand, we analyzed the co-localization of the two Golgi-markers under different experimental conditions. Relevant cells (Figure 3) were selected out of hundreds from a widefield screen to be representative and characteristic for the respective condition. The Golgi complex is a rather compact and large structure; the labeled proteins occur in high numbers and were marked with dye conjugated antibodies. We note that the density of fluorophores will be high, so that the amount of photobleaching during widefield screening and confocal imaging will be small and should not affect dSTORM resolution.





Confocal imaging visualized the partially fragmented Golgi complex, which was observed after 20 min incubation with nocodazole, fairly well by imaging either marker (Figure 3C; see also Figure 1A for widefield). ICA of the confocal image revealed not only more co-localization than in the dSTORM images (Table 2) but also a broader distribution of the co-localizing pixels in different cells (71% in confocal instead of 4.2% in dSTORM) (Figure 3C). Furthermore, dSTORM imaging can resolve GM130 and GalT structural motifs that are hidden or unresolved in the confocal mode (Figure 3C, 3F). This allows the collection of more reliable information on size, number, and shape of the Golgi ministacks after treatment of cells with nocodazole.

Table 2. 





Treatment of cells with Brefeldine A (BFA) induces redistribution of the Golgi complex to the endoplasmic reticulum (ER) (Figure 3B, E) (19, 20). In contrast to the experiments with nocodazole, where both GalT and GM130 had a similar localization pattern, GalT and GM130 showed a different behavior after BFA treatment. GalT was re-localized to the ER and formed a more reticular pattern, whereas GM130 appeared at the discrete ER exit sites, perceived as punctuate structures (21-24). Even if the separation of these two localization patterns is easily achieved by a confocal microscope (Figure 3B), dSTORM provides a more refined distribution, size and number of punctuate GM130 specific structures (Figure 3E). Similar to the experiments with nocodazole, a considerably improved co-localization of both markers is obtained (Table 2). The same is true for the untreated cells with an intact Golgi complex (Figure 3A, D). Spatial resolution of cis- and trans-Golgi markers is increased nearly 2-fold according ICA analysis in dSTORM imaging compared with confocal images of the same cells (Table 2).

For the intact Golgi complex, co-localization analysis based on confocal images might be even more difficult due to the high densities of the markers (Figure 3A, G). Clearly, dSTORM in combination with ICA analysis is able to overcome these problems, particularly in the compact regions of the Golgi complex (Figure 3D, J). In addition, single-molecule based super-resolution techniques offer the additional advantage of providing molecular coordinates, which can be further processed in, for example, molecular-level co-localization analysis (13, 14). This is significantly different to co-localization analysis in confocal microscopy, which is based on the intensity information in individual pixels and thus is spatially constrained. In a coordinate-based approach applied to single-molecule localization data, a co-localization value can be attributed to each single molecule.

The correlative microscopy approach presented here combines fast widefield imaging of large sample numbers with high- and super-resolution microscopy, providing near-molecular level information on cellular structures. If certain structures of interest found in a widefield screen need further investigation by techniques with high resolution, the best representative regions can be chosen from the widefield images. The widefield imaging mode is then used as a filter or trigger system in order to identify predefined structures of interest and find the positions that are to be imaged in the other microscopy modes. This would be beneficial in high-throughput screening experiments, where a manual selection of cells for higher resolution imaging becomes quickly impractical due to the huge amount of data. Even in experiments where a sparse phenotype should be relocated and imaged in high-resolution systems, this approach can help by providing an image of the whole sample at low resolution. The high-resolution image can be recorded at exactly the right position by marking the appropriate spot in the low-resolution image. Due to the possibility of performing multiple experiments on the same cell, the information content is expanded, assuming that photobleaching is negligible (e.g., large structures with high concentrations of labeled proteins, short imaging times per region beforehand). Our workflow for relocating cells on different microscopic systems is furthermore not restricted to the fluorescence microscopic methods presented here, but is also applicable for the integration of any microscopic technique where the position of the reference markers can be identified.

Author contributions

The conception of the study, writing, and editing of the manuscript were done by all authors; the development of integrative microscopy approach and assay adaptation were done by B.F., M.G., J.R., T.L, V.S., M.H., and H.E. The study was executed by B.F., M.G., T.L., and J.R., ICA analysis of dSTORM images was performed by B.F.

Acknowledgments

We thank Sebastian Malkusch for help with co-localization analysis. This work was supported by contract research “Methoden für die Lebenswissenschaften” of the Baden-Württemberg Stiftung (grant nr. P-LS-SPII/11) and by the ”New methods in systems biology” program of the Federal Ministry of Education and Research (grant nr. 0315523A). The ViroQuant-CellNetworks RNAi screening facility is supported by the CellNetworks-Cluster of Excellence (EXC81).

Competing interests

The authors declare no competing interests.

Correspondence
Address correspondence to Holger Erfle, BioQuant Centre, Heidelberg University, Heidelberg, Germany, E-mail: [email protected]">[email protected], or Mike Heilemann, Goethe University Frankfurt, Frankfurt, Germany, E-mail: [email protected]">[email protected].

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