Revolutionizing Wet Sample Electron Microscopy with LVEM and Graphene Liquid Cells

An innovative approach to liquid phase electron microscopy examines samples in Graphene Liquid Cells (GLCs) using Low Voltage Electron Microscopy (LVEM). This combined technique allows for the visualization of organic samples in their native hydrated environments. The encapsulation of samples into graphene liquid cells is now an easy-to-implement, automated procedure. Combined with low-voltage electron microscopy, the hydrated samples can be imaged with uniquely high contrast and resolution.

Observing molecular features of organic samples in their hydrated environment with optimal contrast and resolution is an urgent need in the scientific community. Liquid-phase electron microscopy (LPEM) offers the prospect of high-resolution data as well as real-time visualization of dynamic processes in liquid [1]. The key limitation in attaining higher resolution is the low contrast of organic materials as well as their sensitivity to the electron beam. LVEM addresses these limitations by operating at voltages below 30 kV. This lower accelerating energy enables the study of low-atomic-weight materials with enhanced contrast while preserving the samples integrity [2].

Until recently, observing wet samples using LVEM had been an obstacle, as conventional wet-cell devices available for imaging of liquid-phase samples in transmission electron microscopy have a total thickness (far) exceeding tens of nanometers. Such devices are opaque to the electron beam at these lower operating voltages. The use of Graphene Liquid Cells (GLCs) under LVEM is set to resolve this limitation.

• The ultrahigh vacuum of the electron microscope prohibits imaging hydrated biological and other wet specimens.

• Organic samples consist mainly of low atomic number elements, resulting in images with low contrast.
• Organic samples are not electrically conductive and are easily damaged by the electron beam.

For decades, staining has been used to increase the durability and contrast of organic samples under the electron beam. However, staining alters the sample (e.g. by a change in pH) and in many cases introduces imaging artifacts [3]. Furthermore, the addition of a pretreatment step reduces imaging through-put.

In recent years, silicon nitride (SiN)-based liquid cell holders have emerged as valuable tools in the field of LPEM, enabling researchers to observe dynamic processes within liquid environments in real time [4]. These holders consist of ultra-thin SiN membrane windows that enclose the liquid sample, allowing for the imaging of biological, chemical, and material science specimens.

However, one significant challenge in using SiN liquid cells is the poor resolution primarily due to the electron scattering caused by thickness variations and bulging in the SiN membrane, which in turn reduces image quality. Moreover, SiN liquid cell holders are often designed for specific TEM platforms, limiting their versatility across different microscopy systems.

GLCs consist of a layered structure: starting from the bottom, a TEM grid, covered with a monolayer graphene sheet, acts as a base upon which a droplet containing the particles of interest is deposited. Subsequently, a second monolayer graphene sheet seals the droplet from the top, encapsulating the liquid sample between the two graphene layers [5] (Figure 1).

Furthermore, the π−π stacking between the two graphene membranes confines the liquid sample within numerous pockets throughout the grid. Typical GLCs have a thickness in the tens of nanometers. Along with the thin pocket, the minimal electron beam scattering of the monolayer graphene sheet makes it the most electron-transparent liquid cell for LPEM samples. This significantly improves the contrast and resolution, notably when imaging materials with low atomic numbers, such as biological samples.

Moreover, graphene is highly conductive and a demonstrated radical scavenger [6]. When used as a substrate in TEM imaging, it protects the sample from electron beam-induced damage, including radiolysis, charging, or heating. Graphene is a robust material that can endure the extreme pressure difference between the ultrahigh vacuum column of the microscope and the internal pressure within the GLC pocket. While the preparation of GLCs has in the past been a very challenging manual procedure, the recent development of automation brings GLC preparation available for non-specialized laboratories.

VitroTEM’s Naiad system prepares graphene and assembles GLCs that contain liquid-phase samples on standard TEM grids. GLC grids are ready for imaging in a matter of minutes so that researchers can focus on sample imaging rather than struggle with graphene preparation. The automated GLC assembly produces reliable and reproducible results, providing researchers with unprecedented insights into nanoparticle dynamics.

Various methods exist for the preparation of GLC samples. The predominant technique involves sandwiching water between two TEM grids carrying graphene on a porous support film. However, the use of double grids compromises graphene flexibility, and the supports significantly obstruct the imaging area. Alternative approaches include applying a top graphene layer to a graphene-coated TEM grid either by transferring the layer from below or using the “touch-down” method [5]. The former introduces mechanical stress and has only been demonstrated with multilayer graphene, while the latter involves adding the specimen liquid as a droplet, or spraying micro-droplets onto the graphene layers, which has demonstrated success in yielding intact graphene over a large area.

VitroTEM presents a reliable method for GLC fabrication called Loop-Assisted Transfer (LAT) [5], utilizing a copper substrate for chemical vapor deposition to grow a single layer of graphene. The copper substrate is then etched to produce a freely floating single-layer graphene on water. GLCs are constructed on a TEM grid with a porous polystyrene film supporting the bottom graphene sheet. The transfer process involves using a metal loop to transport both a water droplet and a pristine graphene sheet onto the grid, resulting in minimal stress and a substantial area of intact graphene in the liquid cells.

Delong Instruments’ LVEM 25E is a low-voltage electron microscope equipped with Transmission Electron Microscopy (TEM), Scanning Transmission Electron Microscopy (STEM), Scanning Electron Microscopy (SEM), Electron Diffraction (ED), and Energy Disperse X-ray Spectroscopy (EDS). This compact instrument produces images with unmatched contrast especially for biologic and light-material samples, with resolution as high as 1.0 nm. Moreover, the lack of special facility requirements, such as cooling, special power, or anti-vibration isolation, makes this instrument a great addition to any biological, chemical, or materials laboratory.

Furthermore, the LVEM 25E is a perfectly suited instrument when it comes to imaging organic materials. It provides well-contrasted and highly detailed images from samples where image contrast is often challenging to obtain. In addition, the system’s versatility maintains the ability to image conventionally prepared samples without any adaptation to sample preparation protocols, and it has the unique ability to image both stained and unstained samples with comparable image quality. Until recently, achieving sufficient contrast for the proper visualization of a specimen’s features has been a major challenge in the electron microscopy of biological or low atomic number materials encapsulated in GLCs. Combining LVEM and GLCs has shown a great potential to tackle this obstacle. When analyzed with the LVEM 25, operating in TEM mode at 25kV, the GLCs showed very promising results:

• Significantly higher image contrast — with the lower accelerating voltage leading to increased electron scattering in lightweight elements.

• Ability to image unstained organic materials — the improved contrast compensates for the traditionally low contrast of organic materials in conventional, high-voltage TEMs.

• Longer lifetime of the GLCs — the lower beam energy inflicts less damage on the liquid cell, allowing researchers more time to analyze the specimen without compromising quality.

In the following case studies, GLCs prepared by VitroTEM have successfully been imaged using Delong Instruments’ LVEM 25, operating at 25kV in TEM mode, to observe various hydrated samples in transmission mode.

Ferritin Protein

The ferritin protein sample used in this study was purchased from Sigma Aldrich with an initial concentration of ≤125 mg/ml in PBS buffer. The final solution for GLC sample preparation was formulated by diluting the initial solution in the same buffer to achieve a particle concentration of 0.8 mg/ml. The GLC’s were imaged using an LVEM 25 in TEM mode at 25 kV, and STEM mode at 15 kV. The dense iron cores of the ferritin particles were readily distinguishable, similar to the image results from conventional TEMs at higher voltages (Figure 2). However, the added advantage of using lower voltages comes into play in demonstrating lighter elements, such as the protein surroundings of the iron cores. The particles were uniformly spread out across the entire grid, with the contrasted areas around the particles highlighting the presence of their liquid surroundings. It is noteworthy that liquid pockets form randomly across the span of the grid, some even with no particles entrapped.

In addition to the ferritin protein sample, the enhanced contrast offered by LVEM provided detailed images of the graphene sheets which make up the GLC, especially in areas where there was wrinkling or stacking of multiple sheets (Figure 3). Another key advantage of observing GLCs with the LVEM25 is its ability to demonstrate dynamic processes within the cells. One such process is radiolysis, a phenomenon critically observed in the context of liquid-phase electron microscopy.

Radiolysis occurs as the electron beam breaks up the water molecule [7]. This is evident by the creation of H2 bubbles [8]. The generated H2 molecules lead into the formation and growth of bubbles, moving around within the cell due to diffusion, Brownian motion, etc., and will eventually disappear [2,7]. Their vanishing can be explained by the reverse reaction as there is ultimately an equilibrium within the cell. The LVEM 25 enables researchers to study the nucleation and growth of these bubbles, a task that is extremely difficult, if not impossible, using conventional high-voltage microscopes (Figure 4). The higher acceleration voltage of conventional TEMs cannot provide the necessary contrast to properly observe lightweight materials, let alone such gas bubbles entrapped between carbon layers. Furthermore, when the area of interest is irradiated with a higher-energy electron beam, the bubble formation occurs at accelerated speeds, making it undetectable in real time.

The gold nanoparticles (50nm) were obtained from Sigma Aldrich. The particles were stabilized in a citrate buffer and the final solution for GLC sample preparation was prepared by diluting the gold nanoparticles to a final concentration of 1 mg/ml. Imaging of gold nanoparticles using the LVEM 25 in TEM mode was done with ease. Notably, the instrument’s high contrast enabled the clear visualization of liquid pockets that are entrapped between the layers of graphene, as illustrated in Figure 5. The presence of the numerous GLC pockets spreading throughout the TEM grid underscores their robustness under electron beam bombardment without being ruptured or burst.

On a different note, to further elaborate on the role of LVEM in achieving high contrast with GLC-contained samples, Figure 6 serves as an excellent example. It effectively illustrates the contrast differentiation among various objects in the field, arranged in an ascending order of brightness: gold nanoparticles appear the darkest, liquid pockets as dark gray, graphene layers in lighter gray, and hydrogen bubbles appear white.

The use of low voltage electron microscopy (LVEM) for the imaging of graphene liquid cells (GLC) offers a groundbreaking solution to the challenges of liquid-phase electron microscopy (LPEM). VitroTEM’s automated GLC preparation system, employing the loop-assisted transfer (LAT) method, ensures reproducible results with high yield and substantial imaging area. Delong Instruments’ LVEM25, operating at 25kV in TEM mode, enables imaging of these GLCs with high contrast and reduced beam damage to light element materials. The collaborative application of LVEM and GLCs demonstrates advantages such as higher contrast, extended GLC longevity, and the elimination of staining requirements.

Successful case studies, including the imaging of ferritin and gold nanoparticles, highlight the potential of using LVEM together with GLCs to obtain unprecedented insights into the structure and dynamics of liquid-phase samples. This approach holds promise for diverse applications, offering enhanced resolution in observing hydrated organic or low atomic number specimens. The breakthroughs presented here pave the way for advancements in liquid-phase electron microscopy, providing researchers with powerful tools to explore the intricacies of biological, chemical, and materials systems in their natural hydrated states. As this technology evolves, LVEM and GLCs are poised to become indispensable tools, unravelling nanoscale microscopic mysteries in liquid environments.

[1] Wu, Hanglong, et al. “Liquid‐phase electron microscopy for soft matter science and biology.” Advanced materials 32.25 (2020): 2001582.

[2] Park, Jungjae, et al. “Graphene liquid cell electron microscopy: Progress, applications, and perspectives.” ACS nano 15.1 (2021): 288-308.

[3] Zhang, Y., et al. “Quantitating Morphological Changes in Biological Samples During Scanning Electron Microscopy Sample Preparation with Correlative Super-Resolution Microscopy.” PLOS ONE, 2017

[4] Pu, Shengda, Chen Gong, and Alex W. Robertson. “Liquid cell transmission electron microscopy and its applications.” Royal Society open science 7.1 (2020): 191204.

[5] van Deursen, Pauline MG, et al. “Graphene liquid cells assembled through loop‐assisted transfer method and located with correlated light‐electron microscopy.” Advanced Functional Materials 30.11 (2020): 1904468.

[6] Textor, Martin, and Niels de Jonge. “Strategies for preparing graphene liquid cells for transmission electron microscopy.” Nano letters 18.6 (2018): 3313-3321.

[7] Schneider, Nicholas M., et al. “Electron–water interactions and implications for liquid cell electron microscopy.” The Journal of Physical Chemistry C 118.38 (2014): 22373-22382.

[8] Grogan, Joseph M., et al. “Bubble and pattern formation in liquid induced by an electron beam.” Nano letters 14.1 (2014): 359-364.

You can view and cite the original article here: MRS Advances

Emad Shahnam, MASc, is an Applications Engineer at Delong America. His experience, notably at the University of Waterloo’s Center for Advanced Materials Joining (CAMJ), involved the development of nanocomposite filaments for water purification, the characterization of organic materials for electroclassification in food science, and the preparation and analysis of Pt/C catalysts for hydrogen fuel cells. At Delong America, Emad develops applications where Low Voltage Electron Microscopy can provide researchers with key insights into their samples.

Sina Sadighikia, PhD, is a seasoned electron microscopy professional with over a decade of experience. He earned his Ph.D. in Liquid Cell Electron Microscopy from Utrecht University and completed a postdoctoral stint at Technical University of Delft before joining VitroTEM as the Lead Application Scientist. In this role, he plays a pivotal part in driving forward the development of groundbreaking electron microscopy techniques and methodologies. Currently, Sina’s primary focus lies in the development of the graphene liquid cell technique for biomaterials, a field with immense potential for transformative impact., Sina’s expertise and dedication to innovation have positioned him as a respected figure in the field, driving forward transformative advancements in electron microscopy.

Learn more about VitroTEM on their website:

https://vitrotem.com/