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Fluorescence detection of protein clusters in individual cells and tissue sections by using toponome imaging system: sample preparation and measuring procedures

Abstract

This protocol details sample preparation and measurement procedures for a fluorescence technology capable of colocalizing hundreds of different proteins in a cell or tissue section. The procedure relies on fixation of samples and on the use of dye-conjugated tag libraries. To colocalize proteins, a sample is placed on the microscope stage of an imaging system (toponome imaging system (TIS)) performing sequential cycles of tag-dye incubation, imaging and bleaching to generate images for each localization cycle. TIS overcomes the spectral limitations of traditional fluorescence microscopy. Image processing reveals toponome maps, uncovering the coexistence of proteins at a location (protein clusters). The approach provides direct insight into the topological organization of proteins on a proteomic scale for the first time. If, for example, two dyes are used per cycle, 18 proteins in 4 visual fields can be colocalized in 21 h. Parallel TIS procedures using more than two dyes per cycle enhance the throughput.

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Figure 1: Schematic illustration of a TIS.
Figure 2: Illustration of the topological hierarchies of proteins within the toponome.
Figure 3: Example of PBMC TIS data acquisition and of the construction of toponome maps at low resolution (overview magnification).
Figure 4: 'Landscape' of protein microclusters on PBMC surface (3D toponome fingerprinting).

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Acknowledgements

This research was supported by DFG Schu627, DFG Innovationskolleg INK15, the Land Saxony Anhalt and the BMBF through projects Biochance and CELLECT, NGFN2 and NBL3. We thank P. Benedix and R. Hillert for the development of new software programs.

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Correspondence to Walter Schubert.

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Supplementary information

Supplementary Figure 1

Toponome mapping of cultured rhabdomyosarcoma cells (PDF 2784 kb)

Supplementary Figure 2

Toponome mapping of a tissue section across prostate cancer tissue (PDF 4599 kb)

Supplementary Figure 3

Non-automated manually controlled imaging cycles on a muscle tissue (PDF 3371 kb)

Supplementary Figure 4

Automated CMP detection (PDF 2317 kb)

Supplementary Figure 5

Expert based CMP detection (PDF 2042 kb)

Supplementary Table 1

Specification of tag library (PDF 27 kb)

Supplementary Video 1

Illustration of the operations of a TIS (MOV 7678 kb)

Supplementary Video 2

Screen shot video. This video illustrates the sequential collection of the 30 most frequent CMPs (out of 9,646 in total) based on automated threshold setting shown in Figure 3b. (MOV 12751 kb)

Supplementary Video 3

3D rocking video of PBMC. This video illustrates the toponome colocalization map shown in Figure 4a (MOV 5716 kb)

Supplementary Video 4

3D rocking video of a single lymphocyte. This video illustrates the toponome colocalization map of a CD4 T-lymphocyte shown in Figure 4b. Note the different cell surface protein clusters displayed in different colours illustrating the cell surface “landscape” (MOV 5148 kb)

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Friedenberger, M., Bode, M., Krusche, A. et al. Fluorescence detection of protein clusters in individual cells and tissue sections by using toponome imaging system: sample preparation and measuring procedures. Nat Protoc 2, 2285–2294 (2007). https://doi.org/10.1038/nprot.2007.320

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