ITO Substrates for CLEM of Fibril Proteins

Tips & Tricks for Scanning Electron and Fluorescence Microscopy

  • Fig. 1: SE-SEM images, left panel: commercial glass+ITO substrate; middle panel: glass+ITO deposited by MS and annealed at 300°C (inset: as-deposited film); right panel: glass+ITO deposited by IBC and annealed at 300°C (inset: as-deposited film). Scale bar is 500 nm for all images and insets.Fig. 1: SE-SEM images, left panel: commercial glass+ITO substrate; middle panel: glass+ITO deposited by MS and annealed at 300°C (inset: as-deposited film); right panel: glass+ITO deposited by IBC and annealed at 300°C (inset: as-deposited film). Scale bar is 500 nm for all images and insets.
  • Fig. 1: SE-SEM images, left panel: commercial glass+ITO substrate; middle panel: glass+ITO deposited by MS and annealed at 300°C (inset: as-deposited film); right panel: glass+ITO deposited by IBC and annealed at 300°C (inset: as-deposited film). Scale bar is 500 nm for all images and insets.
  • Fig. 2: Optical transmittance spectra in the 500-900 nm wavelength range of the following substrates, left panel: only glass; commercial glass+ITO; glass+ITO as-deposited by MS; glass+ITO both as-deposited by IBC and annealed at 250 °C. Right panel: only glass; commercial glass+ITO; glass+ITO deposited by MS and annealed at 300° C; glass+ITO deposited by IBC and annealed at 300°C.
  • Fig. 3: CLEM images of A568 labelled α-Syn fibrils placed on the commercial glass+ITO substrate (left panel), MS as-deposited glass+ITO (middle panel) and IBC as-deposited glass+ITO (right panel). FM and SE-SEM images were acquired with the same microscopes’ settings for all substrates. Optical images were acquired by means of a 60x/1.4NA oil immersion objective, exciting the AF568 by the 561/14 nm wavelengths of a LED light source (power: 200 mW). The emitted fluorescence was collected in the bandwidth 607/34 nm by a sCMOS camera. The SE-SEM images were acquired by using an Everhart-Thornley in-chamber detector, in a SEM equipped with a Schottky FEG electron source, working at 30kV of acceleration voltage, and keeping constant the beam current. Scale bars in all images: 1 μm
Correlative light and electron microscopy in a SEM is usually performed using glass commercial substrates coated with indium-thin oxide. These substrates however show a patchy background unavoidably imaged when fibril proteins are deposited on them. To avoid this effect, keeping both optical transparency and electrical conductivity of the same kind of substrates, we propose two cheap and easy preparation methods by either magnetron sputtering or ion beam coating.
Conventional fluorescence microscopy (FM) is capable of imaging specific proteins by using fluorescent labels purposely targeting them. As well, it can reveal their presence and localization in living cells. However, the typical resolution of conventional FM, as stated by the Abbe’s rule, cannot be better than few hundreds of nanometers. Conversely, electron microscopy (EM) demonstrates its capability to either directly image sole proteins or indirectly localize their presence in cells and tissues down to nanometer scale. Thus, combining the information provided by the FM and EM from the same zone of the specimen consents to localize the protein(s) of interest, as provided by FM, and then to refine the investigation with the resolution typical of EM. Such a combination is usually referred as correlative light and electron microscopy (CLEM), where the EM can be performed either in transmission (TEM) or scanning (SEM) mode. In the last years, commercial devices have allowed performing FM inside a SEM, aiming at imaging a given region of sample containing fluorescent proteins first by FM, then, by a proper alignment procedure, the same zone by SEM, hence with higher magnification/resolution and under the vacuum conditions the microscope is capable to provide. Thus, the images obtained by the two techniques can be finally merged and correlated. This approach has been shown to be effective for imaging both sole proteins deposited on a substrate or proteins contained in thin slices of cells/tissues, in the latter case with the main advantage of avoiding to label them by gold nanoparticles. However, in order to perform CLEM experiments, the sample has to be placed on a substrate allowing to image it by both FM and SEM, meaning that it has to be concomitantly transparent for the used light wavelengths and electrically conductive.

Typically, such a substrate is constituted by glass covered by a thin film of tin-doped indium oxide (In2O3 90%, SnO2 10%), commonly called ITO.

CLEM of Amyloid Fibrils
We performed CLEM of amyloid fibrils formed by the human α-Synuclein (α-Syn) proteins labelled with AlexaFluor 568 (AF568) by a FM-SEM dedicated device (Secom platform, Delmic, integrated in a Hitachi SU5000 SEM), using expensive, commercially provided substrates constituted by glass covered with an ITO film with not specified thickness. As shown in figure 1, left panel, the SE(Secondary Electrons)-SEM image clearly displays a patchy texture due to the presence of several crystal ITO domains with random orientation and then different electron channeling effect. If on one hand such a texture is usually not visible when CLEM is carried out on thin slices of resin embedding cells or tissue placed on the ITO-coated commercial substrates, on the other hand when the same imaging is performed on α-Syn fibrils here reported in figure 3, it results in an unavoidably inhomogeneous background contribution (fig. 3, left panel), compromising the image quality. Thus, with the aim to avoid this effect, we prepared new substrates depositing 20 nm of ITO on previously cleaned glass coverslip by both magnetron sputtering (MS, using a Safematic CCU-010 HV sputter coater working with a coating current of 60 mA and an Ar pressure of 5x10-3 mbar) and ion-beam coating (Gatan IBC, using 4keV of Argon ion-beam energy, and rotating the sample at 60 rpm). Once the ITO films were deposited, the two as-prepared different substrates were first examined in terms of SE-SEM imaging and optical transmittance, the latter for wavelengths ranging from 500 to 900 nm and normalized to the lamp intensity. Both as-deposited ITO films did not show any patchy network in the SE-SEM observation as displayed in the insets of figure 1 (middle and right panels), thereby indicating that they were likely still in the amorphous state. However, as shown in figure 2 (left panel) the transmittance of the IBC as-deposited films was lower than that of the MS one, being this effect more pronounced at the lower wavelengths. For further comparison the transmittance spectra of both the only glass coverslip used as substrate to deposit ITO, and the commercial Glass+ITO substrate are also reported in the same graph. The optical properties of the Glass+ITO substrate obtained by IBC deposition change once it is annealed for 9 hours under 0.1 bar of N2: at 250°C it does not show any trace of patchy texture (SEM image not shown), but its optical transmittance becomes analogous to that of the film as-deposited by MS (fig. 2, left panel). However, if annealed at 300°C, a further increase of the light transmittance is observed only for the film deposited by MS (fig. 2, right panel), and not for that deposited by IBC. Besides, as shown in the middle and right panel of figure 1, when imaged by SE-SEM both ITO films show the patchy texture related to their crystallization after annealing at 300°C. The patchy texture of commercial Glass+ITO substrate is also visible in figure 3, left panel, where the FM image of the AF568 labelled α-Syn fibrils has been merged with the SE-SEM one of the same sample area. The homogeneous background in the SE-SEM image of both the as-deposited by MS (fig.3, middle panel) and by IBC ITO film (fig. 3, right panel) comes at the expenses of a reduction (low for the former and higher for the latter) of the grey levels of the single fibrils AF568 signal in the fluorescence image, compared to the AF568 signal measured on the commercial Glass+ITO substrate, as expected considering the corresponding light transmittance spectra.
Our results point out that a cheap and easily obtainable alternative to the expensive commercial CLEM substrates could be represented by either Glass+ITO substrates as-deposited by MS or deposited by IBC and then annealed at 250°C under inert atmosphere with low pressure. In both cases their optical transmittance properties are very similar to that of the commercial substrate, but with the further advantage of not showing any patchy background when the samples placed on them are imaged by SE-SEM.  

Simona Rodighiero1, Bruno Torre2, Joakim Reuteler1, Dorothea Pinotsi1, Elisa Sogne2, Andrea Falqui2

1Scientific Center for Optical and Electron Microscopy (ScopeM), ETH Zürich, Switzerland
2King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia

Dr. Simona Rodighiero
European Institute of Oncology
Imaging Unit Coordinator
Milan, Italy

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