FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles

摘要: Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light (LM) with (correlated LM EM: CLEM) find areas interest, the labeling molecules still a challenge. We present new genetically encoded probe for CLEM, named “FLIPPER”, which facilitates quantitative analysis ultrastructural features cells. FLIPPER consists fluorescent protein (cyan, green, orange, or red) visualization, fused peroxidase allowing visualization targets level. The use straightforward because module completely encoded, can be optimally prepared examination. quantify cellular morphology level expressing normal disease-causing point-mutant cell-surface called EpCAM (epithelial cell adhesion molecule). mutant retained endoplasmic reticulum (ER) could therefore alter ER function morphology. To reveal possible alterations, were co-transfected color-coded full-length targeted ER. CLEM mixed population allowed color-based identification, followed an unbiased ultrastructure EM. Thus, combines bright proteins optimized live imaging sensitivity labeling, thereby representing promising tool CLEM.