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

Jeroen Kuipers,Tjakko J Van Ham,Freark Dijk,Anneke Veenstra-Algra,Ruby D Kalicharan,Ben N G Giepmans,Klaas A Sjollema,Ulrike Schnell

doi:10.1007/s00441-015-2142-7

Abstract

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named “FLIPPER”, which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-015-2142-7) contains supplementary material, which is available to authorized users.

Highlights

The study of the dynamics of molecules in living cells and the determination of their location at the ultrastructural level areCell Tissue Res (2015) 360:61–70 important for understanding the molecular mechanisms underlying cell behavior
FLIPPERs were fused to a part of mannosidase II; this ensured Golgi localization, as successfully applied previously with the tetracysteine system (Gaietta et al 2006) and wellproven targeting for the endoplasmic reticulum (ER) based on calreticulin and an Nterminal KDEL sequence (Fig. 1a)
The majority of genetically encoded probes that can be detected at both light microscopy (LM) and Electron microscopy (EM) levels are based on continuously improving fluorescent proteins and/ or osmiophilic DAB precipitates (Table 1)

Summary

Introduction

The study of the dynamics of molecules in living cells and the determination of their location at the ultrastructural level areCell Tissue Res (2015) 360:61–70 important for understanding the molecular mechanisms underlying cell behavior. Encoded fluorescent proteins (FPs) allow the imaging of molecules and organelles in living cells (Shaner et al 2007) and fluorescent-based microscopic techniques have been developed that permit near-molecularresolution imaging of biomolecules (Schermelleh et al 2010). To identify molecules by CLEM, dedicated probes have been designed that give spatiotemporal information in living cells but that can be detected in their well-preserved ultrastructural surroundings by EM These probes should combine the benefits of (1) the ability to use fluorescence in live cells and (2) their visualization by EM without destructive treatment of cellular membranes

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Conclusion