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High-resolution imaging with field-emission SEM: the need for downstream plasma cleaning in biological applicationsLydia-Marie JoubertCell Sciences Imaging Facility, Beckman Center, Stanford University School of Medicine, Stanford, CA, USA

IntroductionThe initial steps in the process of high-resolution scanning electron microscopy (SEM) of biological samples are well described [1, 2] and involve various steps of aldehyde fixation and dehydration, followed by critical point drying with liquid CO2, or chemical drying with hexamethyldisilazane, or cryo-fixation and lyophilization. In all cases the samples need to be mounted onto an instrument-compatible platform (i.e. the specimen stub) with a conductive adhesive layer, which may consist of carbon or copper tape, colloidal graphite or silver paint. Finally the specimen is coated with a thin conductive film of metal, typically gold and/or palladium, iridium, or platinum. In cases where heavy metals may interfere with the secondary electron (SE) or backscattered electron (BSE) signals needed for the detection of valuable information, carbon evaporation can be used to provide a conductive coating on the sample.

“Cleanliness is next to godliness” Precision measurements, unobscured visualization and limitation of imaging artifacts are the final steps challenging high-resolution field-emission scanning electron microscopy (FE-SEM) of biological samples.

Recent novel developments in SEM column and lens manufacturing, beam control and aberration correction, as well as detector design and high-performance electron guns, have enabled high resolution, high magnification imaging at low accelerating voltages, thereby limiting charging of samples and introduction of beam damage to delicate samples [3]. The benefits of a low-voltage beam mean that the conductive sputter-coated layer can now be limited to a few Angstroms, in comparison to the 100-200Å previously needed to avoid sample charging [4]. Consequently, on samples with limited conductivity, the signal-to-noise ratio (SNR) cannot be improved by increasing the accelerating voltage when beam perturbations occur, as this will result in increased charging and beam damage. Thus, to achieve high resolution visualization and image capture, the cleanliness of the sample, specimen chamber and electron column, have become important aspects to ensure signal stability and high SNR during image capturing.

The importance of SNR is well-known,

although the factors contributing to its parameters are not always well understood. Noise has been described as the single most important limiting factor in scanning electron microscopy [3] and although it is by definition an unwanted aspect that intrudes into the image, it cannot always be eliminated. In the SEM environment, noise results from fluctuations in the signal from a particular pixel, even when incident beam, sample and recording conditions are kept constant. While electron production from the cathode and electron interaction with the specimen are statistical in nature and an integral property of the signal, the electron microscopist needs to optimize conditions and maximize SNR to obtain high-resolution images. Attempts to maximize SNR through the adjustment of parameters such as beam voltage, aperture size, working distance, scan speed and noise reduction algorithms, has revealed the importance of a clean imaging environment throughout the SEM, including the prepared and mounted specimen.

plasma cleaningFor SEM users to obtain high-quality, repeatable and quantitative measurement results, dedicated research and collaboration with industry have led

to the development of plasma-cleaning devices and procedures [5] (Figure 1).

During downstream plasma cleaning, the SEM specimen chamber is flooded with oxygen radicals that oxidize carbon compounds which are consequently removed by the instrument’s vacuum pumps. The RF plasma generator and RF electrode uses air to produce oxygen radicals at optimal gas pressure, frequency and power. Hydrogen, helium and argon have also been introduced when required by, and compatible with, sample, instrument and environment. Passive plasma cleaning, in comparison to active cleaning [6], does not introduce the treated material as a current collecting electrode, surface temperature remains low and adherent layers of hydrocarbons as well as chemical contaminants are removed from the treated surface. Fresh gas is continuously fed to the system while contaminated species are sucked away. It is important to experiment with the characteristic parameters to optimize the cleaning rate and efficiency.

The real battle against SEM contamination started in the 1990s, and was initially focused on the integrated circuit industry, where an estimated 1 nm improvement in IC technology

Figure 1 Principle of plasma cleaning in a scanning electron microscope.A radio frequency-generated plasma is contained in the module, producing reactive gas radicals which are drawn through the vacuum chamber where they breakdown unwanted hydrocarbons into smaller constituents which are easily removed by the microscope’s vacuum system.

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may have an estimated 1 billion dollar economic impact [7, 8]. This has become so important that NIST specifications require regular monitoring of contamination performance and separation of sample-related from instrument-related contamination [9]. Reliable and repeatable performance has become equally important in biological applications, where accuracy is needed to provide information on metabolic functions, spatial relationships and micro-elements, which translates to applications in pharmacy, medical devices, and health and environmental control.

The recent and continued development of novel 3D serial SEM techniques of resin-embedded specimens using BSE detection [10, 11], with continuous high accelerating voltage scanning of surfaces at short working distances introduced a range of contaminants into the specimen chamber and upper and lower column of our Zeiss Sigma SEM, with detrimental effects not only for continued BSE imaging, but also for conventional Everhart-Thornley and In-Lens (aka Through-Lens, TLD) SE imaging. Not only was the SNR markedly affected by outgassing resins and adhesive tape, but apertures were coated and clogged with vaporized polymeric materials, leading to a loss in signal and a rapid increase in noise levels. Contaminants in the specimen chamber and electron column further caused beam instabilities which were visible especially during extended scan cycle times needed for high resolution imaging. Additionally, exploration of low-conductivity and beam-sensitive samples challenged the SNR at the sample-compatible voltages.

In this article we report on our exploration of not only the value of proper processing, handling and mounting of samples, but also the effect of regular downstream plasma cleaning of FE-SEM and biological samples.

Methods

SAMPLE PREPARATION FOR SEM Materials for conventional SEM imaging were prepared with generic methods of glutaraldehyde or paraformaldehyde fixation, OsO4 post-fixation, stepwise ethanol dehydration, and critical point drying. Dried samples were mounted on aluminum stubs of appropriate size, and sputter-coated with a thin (40-70Å) layer of Au/Pd.

For serial sections, samples were fixed and dehydrated similarly, followed by gradual infiltration with EMBed 812 (Epon) epoxy resin (Electron Microscopy Sciences, Hatfield, PA, USA), and polymerization at 60°C. Serial sections were cut with a Leica Microsystems EM UC6 ultramicrotome and collected on gelatin-subbed coverslips, followed by staining in uranyl acetate (3.5%, 20 min) and Reynold’s lead citrate (1%, 2 min), carbon evaporation and mounting with colloidal graphite on 50 mm Al stubs.

Samples were examined in a Zeiss Sigma field-emission scanning electron microscope.

Plasma cleaningThe SEM specimen chamber was plasma cleaned with an Evactron De-Contaminator (XEI Scientific, Redwood City, CA) at 400 mTorr, 20 W for 6 minutes daily after imaging. Specimens were additionally cleaned in-situ in the SEM chamber

Figure 2 Effect of contamination in scanning electron microscope images of biological samples. (A) Objective aperture removed due to extremely poor SNR: SEM image reveals contamination and clogging of 30 µm aperture. (B) Immunogold labeled Helicobacter pylori: beam instability during image capturing. (C) Helicobacter pylori on poly-L-lysine-coated glass coverslip, fully processed and Au/Pd coated: the darkened area on the bacterial sample results from hydrocarbon accumulation. .

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Figure 3 Images of ultrathin (100 nm) sections of an Epon-embedded sample of Helicobacter pylori on Caco-2 cells obtained before (A) and after (B) plasma cleaning (3 cycles, 6 minutes each).

at 100 mTorr, 15 W for 3-4 minutes as needed and allowed by specimen properties. Results and Discussion:In our high-resolution biological FE-SEM applications, contamination was seen as: · material build-up and clogging of apertures (Figure 2A)· increased noise levels, and therefore low SNR (Figure 3A)· low contrast (Figures 3A and 4A)· loss of spatial resolution, with a lack of ultrastructural details – i.e. edges appear smeared (Figures 3A and 4A)· beam deflection during slow scan speeds and high resolution image capturing (Figure 2B)· a dark rectangle from focused frame (Figure 2C)

With the increase in surface resolution that new the InLens detectors enable, the familiar and generally recommended 100Å conductive coating of biological (and non-conductive) surfaces is revealed as a granular coating, and has become intrusive to sub-micrometer and nanoscale features, which effectively masks the real surface details. Application of low-voltage SEM proved to be increasingly beneficial for cases where conductivity was limited by a thin 20-30Å gold/palladium coating to reveal the natural ultrastructure. Limiting beam-sample interaction volume through low accelerating voltage (<2 kV) provided specific surface details, but also required a high-vacuum clean environment. In SEM imaging often the contaminants introduced into the system by a previous high-voltage user can interfere with low-voltage imaging of a new sample. Plasma cleaning of the SEM after high voltage cleaning, and especially after imaging of resin-embedded sections, were required before continuing with low voltage scanning.

In the case of immunolocalization with conjugated nanogold (5-20 nm) secondary antibodies, or silver-enhanced nanogold, exact measurement of nanoparticles has become imperative, especially in cases where more than one particle size are used to localize multiple proteins in a single experiment. Since exact measurements are strongly affected by the cleanliness of the vacuum environment, Evactron-cleaning of our specimen chamber was required before visualizing immunolabeled specimens.

The immediate effect on resolution can be determined in the higher SNR obtained using identical imaging parameters. As a result a lower accelerating voltage of 2-3 kV can be used for SE imaging, and 3-6 kV for BSE imaging. This implies that less charging and beam damage are introduced to fragile specimens.

It has become evident that not only is a clean instrument needed to produce accurate and unobstructed results, but also a clean sample, and therefore establishment of a sample-cleaning regimen, is equally important. With the plasma-cleaning unit mounted on the Zeiss Sigma FE-SEM the power and pressure range was adjusted for instrument cleaning (20 W, 400 mTorr) versus sample cleaning (15 W, 100 mTorr). Plasma time varied between 3-6 minutes. Once the specimen chamber, apertures and column have been cleaned, after contaminated apertures were replaced as needed, a 6 min daily plasma-cleaning (RF 20 W, pressure 400 mTor) controlled new

buildup of hydrocarbons, and chemical buildup in the entire system.

Figures 3 and 4 illustrate the improvement in resolution, SNR, beam stability and clean surface for unobtrusive image capturing of images of biological and gold samples.

ConclusionsOnce the FE-SEM chamber and column parts have been cleaned, and cleanliness is maintained through daily plasma cleaning after imaging, overall performance is enhanced, and lower and upper column maintenance is better controlled.

Although plasma cleaning is performed in the specimen chamber only, the efficient removal of contaminants from the chamber area prevents the drift of hydrocarbons and chemicals into the rest of the system where aberrations can manifest as beam arching, aperture closure, drift and low SNR. These problems can then

result in low spatial resolution, poor contrast, imaging artifacts, inaccurate and irreproducible measurements.

The most common indication of the buildup of carbonaceous materials remains the characteristic dark pattern, corresponding to the scanned surface frame. This feature has become such a familiar challenge in SEM imaging, that teaching and training in SEM without exception include reference to the ‘black square’, with recommendation to avoid focusing, and doing beam adjustments on the exact region of interest. In a thoroughly and regularly plasma-cleaned system it may even become challenging to find a blackened area to demonstrate the accumulation of hydrocarbons on a biological sample.

Painstaking dedication to a plasma-cleaning regimen is required to maintain a pristine imaging environment. The benefits, however, are countless, not only in image quality, data

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biography Dr Lydia-Marie Joubert is an SEM specialist at Stanford University’s Cell Sciences Imaging Facility, where she does technique development, training, teaching and user support. She received the MSA Professional Staff Technologist Award (2009) for developing hydrogel imaging techniques with VP-SEM. She holds a PhD in plant sciences from the University of Pretoria, South Africa, and Indiana University, USA (1986), as well as an MSc in botany (1983) and MPhil in higher education (2003) from the University of Stellenbosch, South Africa. She was previously involved in environmental microbiology and bioenergy research.

abstractFor high-resolution visualization and image capturing of biological samples, the cleanliness of the high-vacuum imaging environment, including both instrument and sample surface, have become important aspects to ensure electron beam stability and high signal-to-noise ratios (SNR). Regular downstream plasma cleaning of both SEM chamber and specimen is required to ensure an uncontaminated instrument that can deliver accurate, reliable and repeatable secondary and backscattered electron data. Development of new imaging technologies and sample requirements may provide novel challenges to instrument and electron microscopist. Control of a clean imaging environment through regular plasma cleaning, prevents contamination build-up throughout the SEM, and results in improved beam stability and SNR for high-resolution biological imaging.

acknowledgementsThe Beckman Center is thanked for financial support, colleagues and students for materials and discussion, and Prof. Manuel Amieva for collaboration on H. pylori.Zeiss service and application engineers are acknowledged for excellent support.

Corresponding author details Dr Lydia-Marie Joubert, Cell Sciences Imaging Facility, Beckman Center B001, Stanford University, Stanford, CA 94305-5301, USA. Tel: +1 650 823 1293Email: lydiaj@stanford.eduWebsite: http://microscopy.stanford.edu/

Microscopy and Analysis 27(4):15-20 (AM), May 2013

©2013 John Wiley & Sons, Ltd

Figure 4Images of gold particles on a carbon substrate test sample before (A) and after (B) plasma cleaning.

accuracy and repeatability, but also in saving time and frustration – which ultimately translates to faster generation of reliable data, increase in scientific knowledge and satisfied electron microscopists.

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