Detection of intracellular granules/foci or other local intracellular particles is a valuable tool for a large variety of biological assays.
Using the Hermes imaging system and Athena analysis SW, granules (or other intracellular foci or small particles), nuclei and cells are each segmented for analysis as separate objects based on their size, number and fluorescence intensity.
- Analysis is applicable at various resolutions and magnifications, depending on the size of the granules/foci.
- Data can be obtained for each cell individually and for a whole well.
Proximity ligation assays (PLA) performed with the Hermes WiScan to identify protein-protein interactions
Here, a group of Hermes imaging system users in Stuttgart university used a multilayered, high-throughput, druggable, genome-wide siRNA screening approach to discover novel components specific for the NOD1 pathway and its interaction with Actin. NOD1 is an intracellular pathogen recognition receptor that contributes to the innate anti-bacterial immune responses, adaptive immunity and tissue homeostasis. NOD1-induced signaling relies on Actin remodeling. Using the Athena intracellular granules application, they identified that the protein SSH1 mediates the interactions between Actin and NOD1. Their results were published in PLOS Pathogens.
Figure: In situ proximity ligation assays (PLA) performed with the Hermes WiScan to identify protein-protein interactions via image analysis. Detection of the interaction between GFP-SSH1 (shown in green) and Flag-NOD1 in transiently transfected HeLa cells. Protein–protein interactions are visualized as small, distinct red spots (PLA signals). Quantitation using the Athena(WiSoft automated analysis software) revealed that the area covered by PLA spots per cell was 4-fold higher in cells expressing GFP-SSH1 and Flag-NOD1 than in neighboring GFP-negative cells.
The cofilin phosphatase slingshot homolog 1 (SSH1) links NOD1 signaling to actin remodeling/ Bielig H et al., PLoS Pathog. 2014 Sep 4;10(9):e1004351. doi: 10.1371/journal.ppat.1004351. eCollection 2014 Sep.