ITO Coating for Correlative Microscopy
Tips & Tricks for Scanning Electron and Fluorescence Imaging
Correlative microscopy of immunolabeled cells performed by scanning electron and light microscopy needs a suitable coating. It aims at making the sample surface conductive without masking the backscattered electrons emitted by the high atomic number immunolabeling nanoparticles. Indium-tin oxide (ITO) has been revealed as an effective coating material, being it conductive, stable against oxidation over time and with mean atomic number low enough to not mask the backscattered electrons signal.
CLEM Imaging of Cells
Performing Confocal (CM) and scanning electron (SEM) microscopy imaging on the same biological sample brings to develop a particular case of correlative light and electron microscopy (CLEM). Thus, due to the fact that SEM basically images the sample surface morphology, this kind of CLEM mainly aims to image proteins located on the sample surface. Some tips and tricks to perform CLEM imaging of cells are reported in the following. Here the cells have been first tagged with both fluorophores and 18 nm-sized immunogold particles targeting the same protein, then fixed and imaged by confocal microscopy. After, they have been dehydrated by ethanol series and critical point drying in view of the SEM imaging. In order to provide the cells with a conductive surface capable to ground the electrons hitting them in the SEM, and then to avoid any charging effect, an appropriate coating procedure has then been chosen. The SEM imaging has been performed by concomitantly using the secondary electrons (SE) and the backscattered electrons (BSE) signal. Secondary electrons allow imaging cells surface with its small features, and backscattered electrons permit to detect the location of the immunogold nanoparticles.
Coating of Non-Conductive Samples
Usually, the coating of a not conductive sample in view of SEM imaging is performed using high atomic number (Z) metals like Au, Ir, Pt, Au/Pd, Pt/Pd, etc. However, even if the use of these coating materials is recommended for their high electrical conductivity and capability to produce a good SE signal, then allowing to work at high magnification, they cannot be used for a CLEM approach as they would unavoidably mask the BSE signal emitted by the immunogold markers used to target the protein(s) of interest.
In the past, two elements have been often used to overcome this limitation: carbon (Z=6) or chromium (Z=24), both electrically conductive and with a sufficiently low atomic number to not mask the BSE signal coming from the immunogold markers. Nevertheless, both of them present some respective drawbacks. Carbon produces a poor SE signal and also acts as major source of surface contamination, the latter strongly increasing with magnification. Thus, it is rather unsuitable for high resolution SEM imaging.
Chromium is able to produce adequately thin and conductive films, and it gives rise to a sufficient production of SE. However, exploiting it as a coating agent brings to face its tendency to oxidize quickly and then to decrease the conductivity. This in turn gives rise to a double limitation. First, prior to be sputtered, if kept under air, the chromium target has to be treated in order to remove the chromium oxide that unavoidably forms on its surface, this being a quite time-wasting process. Second, when chromium is deposited on a sample surface, once exposed to the air, it again undergoes oxidation, thus losing its conductivity. In such a case, only redepositing further layers of chromium could make the sample surface conductive again. A smart way to solve all these limitations is using indium-tin-oxide (ITO) as coating material . It shows good electrical conductivity (» 2 ´ 104 Ω-1 cm-1), has a mean Z of 49.1, which is lower enough with respect to the one of the immunogold labelers, acts as a good source of SE, and is stable against oxidation. In  it has also been shown that depositing a 20 nm-thick film of ITO on irregular cells surface, such as that of HeLa (cervical cancer) cells and neurons was sufficient for assuring electrical conductivity, a good SE signal, and not masking the BSE signal emitted by the immunogold markers. The ITO layer can be deposited by using both either a magnetron-based or an ion beam sputter coater, as reported in , with the Ar ions accelerated at a final energy up to 10 keV, corresponding to a deposition rate of 0.14 nm s-1. To coat in the most effective way all the small cell features like filopodia, synapses and dendrites, it has been fundamental to rotate and tilt the sample during the whole ITO deposition. In  the samples have been fast rotated with a speed of 45 rpm and concomitantly tilted on an angular range of 0°-50°, with a speed of 25° s-1.
Figure 1 shows CLEM results reported in  using a SEM operating at an acceleration voltage of 10 kV, and ITO as coating material on HeLa cells. Panel a and b (scale bar: 20µm) show the light microscopy images of a cells group in differential interference contrast (DIC) mode, and a single confocal optical section of the same field of view, respectively. The green color relates to the Alexa488 florescence signal tagging the CD147 protein, highly expressed on the HeLa cells plasma membrane. The white rectangle reported in panel b corresponds to the field of view displayed in panel c (scale bar: 2µm). As well, the area surrounded by the white rectangle in panel c corresponds to the whole field of view shown in panel d (scale bar: 1µm, as in panels e-h), imaged by SEM (SE) after dehydration, critical point drying and ITO coating. The upper rectangle in panel d corresponds to the area that in the fluorescence confocal image (panel c) gave the most intense signal, while the lower one corresponds to an area with quite low fluorescence signal. The upper area is then further magnified and shown in panel e (SEM-SE signal, showing the fine surface morphology) and f (SEM-BSE, showing the compositional contrast). The small white arrows shown in panel f indicate the presence of each immunogold particle, as also highlighted in the inset. As well, the area surrounded by the lower rectangle in panel d is magnified and shown in both panel g (SEM-SE) and h (SEM-BSE). The amount of markers found in both panel f and h indicates the good correlation between the fluorescence observed in panel c and the immunogold markers presence. Overall, these data show that ITO acts as an effective material to perform CLEM, providing good SE signal and electrical conductivity, to locate the immunogold markers and with a very good stability over time in different enviorenments.
 Simona Rodighiero, Bruno Torre, Elisa Sogne, Roberta Ruffilli, Cinzia Cagnoli, Maura Francolini, Enzo Di Fabrizio, and Andrea Falqui: Correlative Scanning Electron and Confocal Microscopy Imaging of Labeled Cells Coated by Indium-Tin Oxide, Micr. Res. and Techn., 78: 433-443 (2015) doi: 10.1002/jemt.22492
Elisa Sogne1, Bruno Torre1, Alberto Casu1, Simona Rodighiero2, Andrea Falqui1
1 King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
2 Scientific Center for Optical and Electron Microscopy (ScopeM), ETH Zurich Hönggerberg, Zurich, Switzerland
Prof. Andrea Falqui
King Abdullah University of Science and Technology (KAUST)
BESE Division, NABLA Lab
Thuwal, Saudi Arabia