SBFSEM is an automated method that can be used to acquire serial images in a SEM for 3D reconstruction made by Denk and Horstman  and then commercialized by Gatan as the 3View® system. The huge volumetric datasets created by this method can be quantitatively assessed at high axial and lateral resolution, as demonstrated by Leapman et al  in their analysis of mitochondrial networks in mouse pancreatic islets of Langerhans. In materials science applications, the SBFSEM technique could be used to create 3D images of bulk materials and interfaces with nanometer resolution via large volumes .
Since hundreds or thousands of images can be present in datasets, loss of image quality due to contamination and charging can ruin an experiment. Moreover, the deteriorating contrast from hydrocarbon contamination makes it necessary to clean or replace the BSE backscatter electron detector frequently.
Remote plasma cleaning with the Evactron® plasma cleaner has been used by many electron microscopists to achieve the best possible images from their instruments by removing or reducing contamination. The oxygen radicals formed in the plasma oxidize hydrocarbon compounds, producing CO2, CO, H2O, and other species which are eliminated by the vacuum system. Various studies conducted at XEI Scientific have demonstrated that the Evactron® plasma cleaner is effective at removing hydrocarbons and this has been proven with quantifiable results applying previously contaminated quartz crystal microbalances to measure the cleaning rates . The present study deals with the benefits of plasma cleaning for optimized generation of 3View datasets and cleaning of backscatter detectors.
Theory and Background
A SBFSEM system comprises of a SEM, a diamond-knife microtome fixed on the inside wall of the microscope’s chamber door, as well as software and hardware to regulate the acquisition process (Denk and Horstmann, 2004). In order to generate serial images without user intervention, a resin-embedded specimen is cut and imaged thousands of times. Figure 1a shows an example of a standard laboratory configuration with the dedicated 3View stage on the FEI Quanta 250 at the Institute of Biotechnology of the University of Helsinki.
In Figure 1b, the close-up of the 3View microtome shows the knife in the clear position on the left and the illuminated sample holder on the right. Depending on port availability, the compact Evactron EP plasma cleaner (Figure 1c) can be attached at several positions on SEM chambers; the port furthest away from the vacuum outlet is the optimal position. Figure 1d illustrates an example of a backscatter detector contaminated with hydrocarbon which showed decreased signal to noise and poor contrast.
Figure 1. Hardware utilized for SBFSEM experiments: a) a typical SBFSEM system, b) details of the Gatan 3View microtome, c) the Evactron EP plasma cleaner mounted on the Zeiss Sigma SEM chamber, d) a contaminated backscatter detector.
Materials and Methods
A Sigma VP SEM (Carl Zeiss Inc.), equipped with an XEI Scientific Evactron EP decontaminator and a 3View SBFSEM System (Gatan Inc.) (Figure 1c), was used. A resin-embedded mouse heart muscle specimen was imaged at 1-3 kV with 2,000X magnification, and the sample was irradiated at 30 kV for 10 minutes. Next, the sample was imaged to check for the formation of contaminated scanning rectangles on the machined aluminum SBFSEM stub and loss of contrast on colloidal silver regions.
Gatan GMS 3.2 software was used to generate histograms of image contrast on sections of Helicobacter pylori on Caco-2 cells and regions of colloidal silver paint before and after cleaning. Using a cleaning recipe of 5 minutes at 20 W, 2 minutes off X5, hydrocarbon contamination from the chamber, sample, and backscatter detector was removed.
In Figure 2, contamination artifacts (a, b) on the aluminum SBFSEM stub is shown which are absent after a single recipe of plasma cleaning (c, d). Figure 3 shows the comparison of adjacent areas of colloidal silver dag, indicating a 14% increase in BSE contrast after a single plasma cleaning cycle. In order to check the consistency of the results, the measurements were repeated three times. A low accelerating voltage of 1.2 kV was then used to make the backscattered electrons more sensitive to a layer of contamination on the detector. When lower accelerating voltages are used, less charging and beam damage are caused to sample surfaces.
Figure 2. Contamination artifacts on the surface of the aluminum stub (a,b) are absent after plasma cleaning (c,d).
Figure 3. A 14% increase in contrast was seen after plasma cleaning a region of colloidal silver dag surrounding the SBFSEM specimen.
Figure 4. The block face of mouse heart tissue was scanned 20X and imaged before and after plasma cleaning.
Figure 5 shows that the results obtained compare favorably with the data published in Joubert . In this example, the examination of contrast levels in both images of resin-embedded cells showed a 15% increase after 3 cycles of cleaning, with each cycle being 6 minutes. Such cleaning protocols allow comparative morphometric analyses and accurate 3D modeling of SBFSEM datasets by enhancing image contrast and SNR, and eliminating charging and imaging artifacts.
Figure 5. Analysis of images in Joubert  indicates a 15% increase in contrast of thin sections of Helicobacter pylori on cACO-2 cells after plasma cleaning.
Besides extending the lifetime of backscatter detectors, the length of the pump down time is dependent on hydrocarbon contamination levels in both FIBs and SEMs. Thus, this time could be utilized as an indicator of the cleanliness of the vacuum system. As shown by the following data, Evactron plasma cleaners not only reduce the pump down time of the FIBs and SEMs but also reduce hydrocarbon contamination. As a result, sample processing throughput is increased without affecting the quality of analysis.
Oxygen radicals are created in remote or downstream plasma cleaning and these fill the vacuum chamber in the form of excited plasma. In Evactron plasma cleaning, air is used as the process gas. At low pressures, a flowing UV afterglow is observed from the excited metastable nitrogen molecules with a characteristic pink/violet color. The concentration of neutral radicals in the plasma flowing UV afterglow is a function of the loss rate in the neutral afterglow and the production rate in the plasma.
Incorporating plasma cleaning into routine operation during SBFSEM experiments can speed pumpdown, reduce charging and contamination artifacts, enhance image quality, and maintain a clean vacuum system during extended data collection. As the need for extended operation in scanning electron microscopes increases, it is necessary to maintain clean conditions in the vacuum chamber. SBFSEM systems should ideally be maintained in uncontaminated condition with uncompromised image quality and must be operational 24/7.
Due to frequent imaging of large blockface resin-embedded specimens, hydrocarbons are released into the vacuum chamber which results in decreased detector efficiency. For instance, to assure best conditions for rapid imaging, the Zeiss MultiSEM 505 is fitted with two plasma cleaners to remove adventitious hydrocarbons in the main chamber and loadlock.
A gentle, down-stream plasma afterglow process is employed by the current generation of Evactron Turbo Plasma™ De-Contaminators to remove hydrocarbon (HC) contamination from various analytical tools, including SEMs and FIBs. At turbo pump pressures, Evactron cleaning speeds up and spreads throughout the chamber. This is because of longer mean-free-paths that cause less recombination of oxygen radicals in the required three body collisions and decreased scattering to chamber walls. In nearly all the cases, short plasma cleaning cycles are adequate to eliminate contamination and drastically decrease pump down time, facilitating high throughput of sample processing and analysis.
The Evactron series of plasma cleaners provides effective, fast, and powerful cleaning over a broad range of pressures, allowing high quality, artifact-free images and increased efficiency of sample analysis. By incorporating plasma cleaning into routine maintenance protocols, optimal detector performance can be maintained.
 Denk, W. and H. Horstmann (2004) PLoS Biol. 2, 1900.
 Leapman, R. et al., (2016) Microscopy and Microanalysis 22 (Suppl. 3), 1104.
 Hashimoto, T. et al., (2016) Ultramicroscopy 163, 6.
 Vane, R and E. Kosmowska (2016) Microscopy and Microanalysis 22 (Suppl. 3), 46.
 Joubert, L. M. (2013) Microscopy and Analysis May Issue, 15.
This information has been sourced, reviewed and adapted from materials provided by XEI Scientific.
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