MICROSCOPY RESEARCH AND TECHNIQUE 78:433–443 (2015)
Correlative Scanning Electron and Confocal Microscopy Imaging of Labeled Cells Coated by Indium-Tin-Oxide SIMONA RODIGHIERO,1 BRUNO TORRE,2 ELISA SOGNE,1,3 ROBERTA RUFFILLI,4 CINZIA CAGNOLI,1 MAURA FRANCOLINI,1,5 ENZO DI FABRIZIO,2 AND ANDREA FALQUI3* 1
Fondazione Filarete, Viale Ortles 22/4, Milano 20139, Italy Physical Sciences and Engineering Division, King Abdullah University for Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia 3 Biological and Environmental Sciences and Engineering Division, King Abdullah University for Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia 4 CEMES/CNRS, 29 Rue Jeanne Marvig BP 94347, 31055 Toulouse Cedex 4, France 5 Department of Medical Biotechnology and Translational Medicine, Universit a Degli Studi Di Milano, Milano 20129, Italy 2
KEY WORDS
scanning electron microscopy; confocal microscopy; correlative microscopy; immunolabeling; ITO
ABSTRACT Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ionsputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. V 2015 Wiley Periodicals, Inc. C
INTRODUCTION Correlating electron microscopy (EM) and confocal microscopy (CM) imaging of cells and tissues is a wellknown method to understand the relationship occurring between cellular structure and function (Agronskaia et al., 2008; Biel et al., 2003; Caplan et al., 2011; Cortese et al., 2009; Mironov and Beznoussenko, 2009). Conventional CM is capable to show where specific molecules of interest are localized, by the use of fluorescent labeling of these molecules. As well, both presence and localization of fluorescent proteins can be visualized by CM in living cells. The typical resolution of conventional CM is predicted by the Abbe’s equation to be few hundreds of nanometers (Abbe, 1873). However, in the recent past, major improvements of the technique have been realized, allowing optical imaging of the observed structures with unprecedented resolution and bringing to the field commonly referred to as “nanoscopy” (Betzig et al., 2006; Gustafsson, 2008; Hell and Stelzer, 1992; Lippincott-Schwartz and Manley, 2009; Schrader and Hell, 1996). On the other hand, EM is still the only imaging technique capable C V
2015 WILEY PERIODICALS, INC.
to directly and routinely look at the cellular ultrastructure down to a nanometer scale that no optical microscope could reach. Combining the information given by the CM and EM on the same area of the specimen allows then to determine the location of the molecule of interest on the cellular ultrastructure. EM imaging could be carried out on biological specimens both in transmission (TEM) and in scanning (SEM) mode. In both the EM approaches, to get information on target distribution, cells can be labeled with probes targeting one or more specific molecules and conjugated with small (
Correlative Scanning Electron and Confocal Microscopy Imaging of Labeled Cells Coated by Indium-Tin-Oxide SIMONA RODIGHIERO,1 BRUNO TORRE,2 ELISA SOGNE,1,3 ROBERTA RUFFILLI,4 CINZIA CAGNOLI,1 MAURA FRANCOLINI,1,5 ENZO DI FABRIZIO,2 AND ANDREA FALQUI3* 1
Fondazione Filarete, Viale Ortles 22/4, Milano 20139, Italy Physical Sciences and Engineering Division, King Abdullah University for Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia 3 Biological and Environmental Sciences and Engineering Division, King Abdullah University for Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia 4 CEMES/CNRS, 29 Rue Jeanne Marvig BP 94347, 31055 Toulouse Cedex 4, France 5 Department of Medical Biotechnology and Translational Medicine, Universit a Degli Studi Di Milano, Milano 20129, Italy 2
KEY WORDS
scanning electron microscopy; confocal microscopy; correlative microscopy; immunolabeling; ITO
ABSTRACT Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ionsputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. V 2015 Wiley Periodicals, Inc. C
INTRODUCTION Correlating electron microscopy (EM) and confocal microscopy (CM) imaging of cells and tissues is a wellknown method to understand the relationship occurring between cellular structure and function (Agronskaia et al., 2008; Biel et al., 2003; Caplan et al., 2011; Cortese et al., 2009; Mironov and Beznoussenko, 2009). Conventional CM is capable to show where specific molecules of interest are localized, by the use of fluorescent labeling of these molecules. As well, both presence and localization of fluorescent proteins can be visualized by CM in living cells. The typical resolution of conventional CM is predicted by the Abbe’s equation to be few hundreds of nanometers (Abbe, 1873). However, in the recent past, major improvements of the technique have been realized, allowing optical imaging of the observed structures with unprecedented resolution and bringing to the field commonly referred to as “nanoscopy” (Betzig et al., 2006; Gustafsson, 2008; Hell and Stelzer, 1992; Lippincott-Schwartz and Manley, 2009; Schrader and Hell, 1996). On the other hand, EM is still the only imaging technique capable C V
2015 WILEY PERIODICALS, INC.
to directly and routinely look at the cellular ultrastructure down to a nanometer scale that no optical microscope could reach. Combining the information given by the CM and EM on the same area of the specimen allows then to determine the location of the molecule of interest on the cellular ultrastructure. EM imaging could be carried out on biological specimens both in transmission (TEM) and in scanning (SEM) mode. In both the EM approaches, to get information on target distribution, cells can be labeled with probes targeting one or more specific molecules and conjugated with small (
