Analyzing proteome topology and function by automated multidimensional fluorescence microscopy

Abstract

Temporal and spatial regulation of proteins contributes to function. We describe a multidimensional microscopic robot technology for high-throughput protein colocalization studies that runs cycles of fluorescence tagging, imaging and bleaching in situ. This technology combines three advances: a fluorescence technique capable of mapping hundreds of different proteins in one tissue section or cell sample; a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors; and a system for imaging the distribution of these protein clusters in a so-called toponome map. By analyzing many cell and tissue types, we show that this approach reveals rules of hierarchical protein network organization, in which the frequency distribution of different protein clusters obeys Zipf's law, and state-specific lead proteins appear to control protein network topology and function. The technology may facilitate the development of diagnostics and targeted therapies.

Opportunities and Challenges in Implementation of Multiparameter Single Cell Analysis Platforms for Clinical Translation

“…9). Extensive literature exists on the development of multiplexed and more recently hyperplexed fluorescence methods for tissue analyses, including instrumentation, software, and bioinformatics tools that can be used in the development of diagnostic tests 9, 20, 21, 22, 23. The ability to measure many parameters in large numbers of tissue samples has propelled these assays into the realm of big data challenges for the future.…”

“…Multiplex protein expression (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) …”

Novel Platforms of Multiplexed Immunofluorescence for Study of Paraffin Tumor Tissues

“…Described previously, the multi-epitope-ligand cartography (MELC) 8,9 , is one of these bleaching techniques, that is capable to map the location of different proteins in one sample of cells or tissues using sequential rounds of fluorescent detection 10 . In each cycle, a couple of antibodies are added; phase contrast and fluorescence images then are acquired by a high-sensitivity "charged Coupled Device", a sensor used in digital cameras to record images; the sample is washed with phosphate buffer saline and bleaches at the excitation wavelengths; and post bleaching phase contrast and fluorescence images are acquired.…”

Automatic Classification of Embryonic Fruit Fly Gene Expression Patterns

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