We present a procedure for real-time imaging and elemental composition analysis of boehmite particles in deionized water by in situ liquid Scanning Electron Microscopy.
In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface (SALVI) and Scanning Electron Microscopy (SEM). This paper describes the method and key steps in integrating the vacuum compatible SAVLI to SEM and obtaining secondary electron (SE) images of particles in liquid in high vacuum. Energy dispersive x-ray spectroscopy (EDX) is used to obtain elemental analysis of particles in liquid and control samples including deionized (DI) water only and an empty channel as well. Synthesized boehmite (AlOOH) particles suspended in liquid are used as a model in the liquid SEM illustration. The results demonstrate that the particles can be imaged in the SE mode with good resolution (i.e., 400 nm). The AlOOH EDX spectrum shows significant signal from the aluminum (Al) when compared with the DI water and the empty channel control. In situ liquid SEM is a powerful technique to study particles in liquid with many exciting applications. This procedure aims to provide technical know-how in order to conduct liquid SEM imaging and EDX analysis using SALVI and to reduce potential pitfalls when using this approach.
Scanning Electron Microscope (SEM) has been widely applied to investigate a variety of specimens by producing high resolution imaging1. The energy dispersive x-ray spectroscopy (EDX) associated with the SEM enables the determination of elemental composition1. Traditionally, SEM is applied for imaging only dry and solid samples. In the last 30 years, Environmental SEM (ESEM) was developed for analyzing the partial hydrated samples in a vapor environment2,3,4,5. However, ESEM is unable to image the wet, fully fluid samples with desired high resolution6. Wet SEM cells were also developed to image wet specimens using SEM7,8; nevertheless, these cells were developed mainly for biological specimens and backscattered electron imaging, and are more accessible for applications with those designs9,10.
To address the challenges in analyzing various samples in their native liquid environment using SEM, we invented a vacuum compatible microfluidic device, System for Analysis at the Liquid Vacuum Interface (SALVI), to enable high spatial resolution secondary electron (SE) imaging and elemental analysis of liquid samples using the high vacuum mode in SEM. This novel technique includes the following unique features: 1) liquid is directly probed in a small aperture of 1 – 2 µm in diameter; 2) liquid is held within the hole by surface tension; and 3) SALVI is portable and can be adapted to more than one analytical platform11,12,13,14,15,16,17,18.
SALVI consists of a 100 nm thick silicon nitride (SiN) membrane and a 200 µm wide microchannel made of polydimethylsiloxane (PDMS) block. The SiN membrane window is applied to seal the microchannel. The fabrication details and key design considerations were detailed in previous papers and patents11,19,20. Currently, a leading manufacturer and distributor of consumable supply for microscopy has purchased the license to sell SALVI devices commercially for liquid SEM applications21,22.
The applications of SALVI in vacuum-based analytical instruments have been demonstrated using a variety of aqueous solutions and complex liquid mixtures including biofilms, mammalian cells, nanoparticles, and electrode materials12,14,17,20,23,24. However, most of the aforementioned work utilized time-of-flight secondary ion mass spectrometry (ToF-SIMS) as the key analysis tool, thus the application of liquid SEM with SALVI has not been explored fully. In this work, SALVI has been used to study larger non-spherical colloidal particles in liquid using liquid SEM imaging and EDX elemental analysis. The sample consists of AlOOH particles synthesized at our laboratory. Submicrometer-sized boehmite particles are known to exist in high-level radioactive waste at the Hanford site. They are slow to dissolve and may cause rheological problems in waste treatment. Therefore, it is important to have the capability to characterize boehmite particles in liquid25. This technical approach can be used to study boehmite in various physicochemical conditions for improved understanding of these particles and related rheological properties. These particles were utilized to demonstrate step-by-step how to apply SALVI to high vacuum SEM in order to study particles suspended in liquid. Key technical points for SALVI and SEM integration and SEM data acquisition are highlighted within the paper.
The protocol provides demonstration of the liquid sample analysis using SALVI and liquid SEM imaging, for those who are interested in utilizing this novel technique in diverse applications of liquid SEM in the future.
1. Prepare AlOOH Liquid Sample
NOTE: Do not touch the specimen or anything inside the SEM chamber with bare hands. Powder free gloves should be worn at all times when handling the SALVI device and mounting it onto the SEM stage in order to avoid potential contamination during surface analysis.
2. Sputter Coat the SALVI SiN Membrane Window with Carbon
3. Mount the Device and Use SEM/Focused Ion Beam (FIB) to Make Apertures on the SALVI SiN Membrane Using FIB
4. Load SALVI with Liquid Samples
5. Conduct Liquid SEM Imaging and Elemental Analysis
6. Plot the EDX Spectrum
The representative results are presented to show how the particles are imaged and analyzed using in situ liquid SEM imaging coupled with EDX. The results include SE images and EDX spectra. The SE images were obtained at 100,000X and 200,000X magnification levels in Figure 1. Figure 1a depicts the SE image of the AlOOH, Figure 1b DI water, and Figure 1c the hole in an empty channel. The images were obtained by applying SE with 8 keV accelerating voltage and 0.47 nA beam current. The screen resolution utilized was 1,024 × 884 with a scan rate of 30 µs. Correspondingly, Figure 2 shows the EDX spectra detected from the AlOOH particles in water (Figure 2a), DI water sample (Figure 2b) and the hole in an empty channel (Figure 2c), based on the measured elemental composition. The EDX spectra were obtained using the same current and voltage setting as that for SE images. The information depth is from the shallow region at the sample surface due to the choice of low voltage. The raw data of the elemental spectra is outputted as.csv file and plotted using a graphing software for presentation.
Figure 1: SE Images. These images were obtained by applying SE with 8 keV accelerating voltage and 0.47 nA beam current. The screen resolution utilized was 1,024 × 884 with a scan rate of 30 µs. (1a) AlOOH at 200,000X, (1b) DI water at 100, 000X (1c) and an empty channel at 200,000X. Please click here to view a larger version of this figure.
Figure 2: EDX Spectra. EDX spectra were acquired in the SE mode with 8 kV accelerating voltage and 0.47 nA beam current. (2a) Spectrum of AlOOH in water. (2b) Spectrum of DI water sample. (c) Spectrum of the hole in an empty channel. Please click here to view a larger version of this figure.
SEM is a powerful technique in surface characterization of organic and inorganic materials on a nanoscale (nm) level with high resolution1. For example, it is widely used for analyzing the solid and dry samples such as geological materials26 and semiconductor27. However, it has limitations in characterizing the wet and liquid samples due to the incompatibility of liquid within the highly vacuumed environment required for electron microscopy1. SEM sample preparation often requires dehydration or freeze-drying for a hydrated specimen, and particularly for biological specimens2. As a result, it is challenging to accurately capture the information of naturally hydrated or liquid samples, as their intrinsic information may be lost during the sample preparation process28,29. This may include but is not limited to, biological activity in cells, synthesis of nanoparticles in solution, aggregation of particles in complex liquid, and electrochemical reactions. Even though the ESEM can image hydrated samples in a controlled vapor environment, the resolution of the images could not reach as high as the SEM images of the solid samples in the high vacuum mode30,31,32,33. Recently, wet samples were covered by an electron transparent thin film6 or sealed by a specimen capsule30 when SEM was employed, and backscattered electrons were collected for images using this approach.
SALVI is a versatile microfluidic interface that has enabled surface analysis of liquids using vacuum-based instruments such as TEM and ToF-SIMS.11,12,13,14 Our technique using SALVI and optimized SEM conditions can provide SE images and EDX compositional information. Figure 1a presents the SE image of boehmite particles in DI water with a submicron scale (400 nm) and high magnification of 200,000. The SEM image shows the morphology and distribution of the boehmite particles in liquid, which validates that the particles in liquid can be seen and held safely within the SiN membrane by surface tension20. In contrast, Figures 1b depicts the SE images of the DI water within the hole at 100,000× magnification level. It provides direct evidence that water can be hold by its surface tension without leaking outside. In addition, the chamber pressure was kept constant at 1.0 × 10-5 Torr during measurement. Figure 1c presents a hole in an empty channel with 200,000× magnification; nothing is observed inside the hole under the same current and voltage settings. The SE liquid imaging capability via this approach provides high resolution SE images compared to the micrometer resolution of backscattered electron images acquired using the reported wet SEM technique30.
EDX elemental mapping is conducted using AlOOH particles in DI water, DI water only, and the empty channel, respectively. The latter two are used as reference controls. As shown in Figure 2a, the aluminum peak occurs at around 1.5 keV with significant signal, while there is no prominent peak appearing at the same energy in the DI water and the empty channel EDX spectra. The oxygen signal is dominant in both AlOOH and DI water, which confirms that this signal comes from water. This further validates that particles are immersed in water during imaging. The C and Si peaks in Figures 2a, 2b and 2c are from the carbon coating on the detection window and SiN membrane forming the detection area, respectively. The N peak is also from the SiN membrane. The EDX comparison shows the detection of the aluminum composition of AlOOH in water, indicating that the boehmite particles are indeed observed.
In previous papers, we have demonstrated the feasibility of employing a microfluidic cell and high vacuum SEM to image and characterize the liquid sample, using DI water and immunoglobulin G (IgG) gold nanoparticles12,20. In these earlier works, gold nanoparticles were smaller than 10 nm. In this work, we show that boehmite particles with much larger sizes (< 100 nm) can also be studied through liquid SEM. The hole size was calculated to ensure sufficient imaging area yet enough surface tension to hold the liquid within. Originally, the hole was fabricated using the gallium ion beam to make round apertures of 2 µm in diameter prior to device assembly in the initial invention12,20. In this update, we show that the detection apertures can be made after the device is assembled, making the entire process more streamlined. One can also open up as many detection windows as needed in an analysis, and is not limited by the holes made before an experiment. The 2 µm diameter detection windows are suitable for techniques such as ToF-SIMS, and it is also feasible in liquid SEM. Because of the high magnification capability of SEM, the new result shows that smaller aperture (e.g., 1 2 µm) works well in SEM (Figure 1a).
Several technical details are worth mentioning in order to make in situ liquid SEM measurements successful. First, the device needs to be coated with carbon or gold in order to reduce charging during the measurements. Second, the device mounting is quite critical in this procedure. Loose contact of the device with the mounting stage will result in significant charging, difficulty in focusing, and poor images. Third, if one wants to analyze more than one sample using the same device, the sample sequence needs some thought. Although the device is disposable, it is likely a device can be used more than once. For example, one can use water or the solvent for obtaining data of the control sample followed by the analysis of a sample with particles or other species of interest using the same solvent. It is recommended to introduce the sample introduction after the SALVI device is secured and detection holes are made using SEM/FIB. The FIB is used solely for milling the holes on the detection window membrane. If the membrane is prepared by another instrument or the membrane is made with holes available from the suppliers, it is not necessary to use FIB to make holes prior to the SEM analysis. Moving the device away from the sample stage for sample introduction and remounting it again wastes a lot of time, while also adding the risk of poor connections between the device and the sample stage and resulting in a different working distance. The SEM operator may also have to refocus and find the channel and micrometer sized round detection windows again.
With submicron resolution and precise elemental information showcased in this study, we envision that the integration of the vacuum-compatible microfluidic cell (i.e., SALVI) with the high vacuum mode SEM can be widely utilized in identifying and observing various naturally hydrated specimens, geological specimens, biological samples, and nanomaterial synthesized in liquid. With the technological improvements made to the liquid SEM approach are discussed previously, we demonstrate that a larger variety of submicron particles of different sizes may be investigated using this new approach. Ultimately, in situ liquid SEM opens more opportunities to study specimens in liquid using high vacuum SEM.
The authors have nothing to disclose.
We are grateful to the Pacific Northwest National Laboratory (PNNL) Nuclear Process Science Initiative (NPSI)-Laboratory Directed Research and Development (LDRD) fund for support. Dr. Sayandev Chatterjee provided the synthesized boehmite particles. Instrumental access was provided through a W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL) General User Proposal. EMSL is a national scientific user facility sponsored by the Office of Biological and Environmental Research (BER) at PNNL. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.
Carbon Coater | Cressington | 208 Carbon | It is accompanied with thickness monitor MTM-10. |
SEM | FEI | Quanta 3D FEG | It provides highly resolved scanning electron microscopy and elemental analysis. |
System for Analysis at the Liquid Vacuum Interface (SALVI) | Pacific Northwest National Laboratory | N/A | SALVI is a unique, vacuum compatible microfluidic cell that enables the characterization of the liquid sample using vacuu- based scientific instrument. |
PEEK Union | Valco | ZU1TPK | The polyether ether ketone union is used for connecting the inlet and outlet of SALVI |
Syringe | BD | 309659 | 1 mL |
Pipette | Thermo Fisher Scientific | 21-377-821 | Range: 100 to 1,000 mL |
Pipette Tip 1 | Neptune | 2112.96.BS | 1,000 µL |
Pipette Tip 2 | Rainin | 17001865 | 20 µL |
Syringe Pump | Harvard Apparatus | 70-2213 | It is used to inject the liquid sample into the SALVI device. |
pH meter | Fisher Scientific/accumet | 13-636-AP72 | It is used for measuring the pH of AlOOH in DI water. |
Barnstead Ultrapure Water System, UV/UF | Thermo Scientific Barnstead | Nanopure diamond D11931 | It is used for producing DI water. |
Centrifuge tubes | Fisher scientific/Falcon | 15-527-90 | 15 mL |
Bransonic ultrasonic cleaner | Sigma-Aldrich | 2510 | It is used to ultrasonicate the AlOOH liquid sample. |
Balance | Mettler Toledo | 11106015 | XS64 |
AlOOH | Pacific Northwest National Laboratory | N/A | It is synthesized by scientists at Pacific Northwest National Laboratory. |
xT microscope Control | FEI | Quanta 3D FEG | Default microscope control software of SEM Quanta 3D FEG |
EDAX Genesis software | EDAX | N/A | The software is used for collecting the EDX elemental information of the samples. |
Teflon tubing | SUPELCO | 58697-U | It is used for introducing the sample into the microchannel and holding adequate volume of liquid. |