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. 2015 Apr;360(1):61-70.
doi: 10.1007/s00441-015-2142-7. Epub 2015 Mar 19.

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

Jeroen Kuipers  1 , Tjakko J van Ham, Ruby D Kalicharan, Anneke Veenstra-Algra, Klaas A Sjollema, Freark Dijk, Ulrike Schnell, Ben N G Giepmans
Affiliations

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

Jeroen Kuipers et al. Cell Tissue Res. 2015 Apr.

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.

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Figures

Fig. 1
Fig. 1
FLIPPER (fluorescent indicator and peroxidase for precipitation with EM resolution), a combinatorial probe for correlated microscopy, combines the advantages of genetically encoded fluorescent proteins (FP) and horseradish peroxidase (HRP) for correlated light microscopy and electron microscopy (CLEM) with high-quality ultrastructural preservation. a Representation of modules emphasizing the straightforward exchange of spectrally different, genetically encoded EM markers (mannII mannosidase II, ER endoplasmic reticulum). b Spectra of FLIPPERs as recorded in transfected cells. The four fluorescent proteins mTurquoise2 (TQ, Golgi), enhanced green fluorescence protein (EGFP; G, ER), mOrange2 (OR, Golgi) and mCherry (R, ER), were excited by using 458-, 488-, 514- and 561-nm lasers, respectively
Fig. 2
Fig. 2
FLIPPER detection by fluorescence microscopy in living cells. Images from living cells taken with a confocal laser scanning microscope. a–d Golgi-FLIPPER based on Turquoise2 (a), EGFP (b), Orange2 (c) and mCherry (d). e–h Secretory FLIPPER based on EGFP (e) and ER-FLIPPERs in the same color-code as in a–d (f–h). Note the typical staining of the Golgi apparatus and ER. Bars 5 μm
Fig. 3
Fig. 3
FLIPPER detection using electron microscopy. The black DAB deposit created by FLIPPER is readily visible in transfected cells but is absent in non-transfected cells. a, a’ Golgi-FLIPPER. Note that not all Golgi stacks are labeled; this can be explained by the localization of mannosidase II to the medial Golgi stacks but not to the cis and trans Golgi (Igdoura et al. 1999). b, b’ ER-FLIPPER. Note the absence of precipitate at nuclear pores and the good preservation of ultrastructure. Membranes are readily visible and mitochondrial cristae are crisp. Bars 5 μm (a, b), 2 μm (a’, b’)
Fig. 4
Fig. 4
Mix and match. FLIPPER in various experimental conditions within a single dish and quantitative EM based on LM. a, b Representative cells expressing (a-a’’) FL (full-length) EpCAM-GFP (epithelial cell adhesion molecule fused to green fluorescent protein) or (b–b’’) mutant EpCAM(C66Y)-mCherry, together with FLIPPER-mOrange2, showing plasma membrane localization of FL EpCAM and ER localization of mutant EpCAM. c–e 293T cells transfected with FL EpCAM-GFP and with EpCAM (C66Y) fused to mCherry and visualized as indicated. f Bar graph indicating thickness of ER in transfected cells as measured by using ER-FLIPPER. n = 100 measurements in 10 cells; error bars indicate standard deviation. Note that the measurements were made under identical conditions in the same experiment. g–i Parts of the boxed cells in c–e. Bars 5 μm (a, b), 100 μm (c–e), 2 μm (g–i)
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