Geckos and Beetles and Ticks! Oh my!

 

"It's always best to start at the beginning - and all you do is follow the Yellow Brick Road."

- Glinda, The Wizard of Oz

    For the second time this week, our nanoscience class embarked on an enchanting journey, following the Yellow Brick Road into the captivating realm of the University of Kentucky's Advanced Science & Technology Commercialization Center, or ASTeCC. Just like Dorothy and her companions in the beloved tale of The Wizard of Oz, we found ourselves in a world of scientific wonders and possibilities. Oh, what an extraordinary adventure it was!

    Our journey began with the environmental scanning electron microscope (ESEM), a powerful imaging tool that operates at different pressures, offering tremendous flexibility in sample analysis. It can function under high vacuum or low vacuum conditions, with the latter being particularly advantageous for biological samples. In the low vacuum mode, the pressure is higher than in the high vacuum mode but still lower than atmospheric pressure. This feature allows for the observation of delicate specimens without the need for extensive sample preparation.

    During our exploration with the ESEM, we had the opportunity to examine various intriguing samples. One captivating specimen was an invasive tick species coated in a thin layer of platinum. By studying the tick's structure and behavior, we aimed to gain insights into effective repellent strategies. Additionally, we observed the head of a sawfly, a coverslip coated in metal and bacteria, a leaf, and even a dandelion seed. These diverse samples showcased the versatility of the ESEM in capturing detailed images across different materials and biological structures.

ESEM image of a tick coated in platinum

Optical Microscope image of the surface of a beetle

    Operating on the principle of a Tungsten electron source pulled down through the column and focused into the chamber, the ESEM employs a magnetic lens to raster the electron beam across the sample. It is important to maintain a distance of at least 10 mm between the stage and the pull piece to ensure proper functionality. However, the high-energy electron beam used in ESEM imaging may cause organic sample materials to degrade. To mitigate sample damage, a sputter coater can apply a protective layer of gold or platinum plasma.

    In our observations, we primarily utilized secondary electron imaging, which provides surface topography information. In this technique, incident electrons dislodge secondary electrons from the sample, which are then detected. Backscatter electron imaging, on the other hand, uses primary electrons that bounce back and are detected. This imaging method offers information about sample density and atomic number, with brighter areas indicating higher density.

    Notably, ticks possess sensors at the end of their legs for detecting carbon dioxide. Analyzing the shapes of their legs through ESEM imaging may provide valuable insights into their sensory capabilities. Furthermore, the ESEM maintains a fixed distance between the sample and the lens. Unlike optical microscopes, where stage or lens movement is employed for focusing, the ESEM achieves focus by altering the lens shape.

    The ESEM's capabilities extend beyond imaging, as it can also perform energy-dispersive spectroscopy (EDS). EDS utilizes X-rays emitted when an electron transitions from an excited state to the ground state, enabling the identification and composition analysis of the emitting atoms in the sample.

    Throughout our exploration, we encountered challenges such as charging effects, where the sample becomes negatively charged by the electron beam, leading to poor resolution and uneven brightness or darkness in the images. To address this, a needle can be introduced to discharge the sample, enhancing resolution. The use of highly conductive coatings like platinum and gold helps prevent sample erosion while maintaining good conductivity.

    The potential applications of an environmental scanning electron microscope are vast and diverse. In biology, ESEM allows for the imaging of living cells and tissues in their natural hydrated state, providing insights into cellular processes, cellular interactions, and the study of microorganisms. ESEM also enables the examination of materials, such as polymers, ceramics, and composites, under various temperature and humidity conditions. This helps in studying material degradation and surface coatings to better understand their properties. ESEM is useful for characterizing nanoscale structures and nanoparticles, providing detailed information on their size, shape, and distribution. Additionally, ESEM helps in the analysis of delicate archaeological and paleontological specimens, providing valuable insights into ancient artifacts, fossils, and historic preservation.

    The Helios Dual-Beam microscope is an advanced instrument that combines a scanning electron microscope (SEM) with a focused ion beam (FIB) functionality. This powerful integration enables comprehensive imaging, analysis, and manipulation of samples at the nanoscale. The FIB component utilizes a focused beam of gallium ions to precisely remove or deposit material from the sample surface. This capability allows for tasks such as milling, creating cross-sections, and fabricating three-dimensional structures with exceptional precision.

    The SEM component of the Helios Dual-Beam microscope functions similarly to a traditional SEM. It employs a beam of electrons to scan the surface of a sample, generating high-resolution images that reveal intricate details of the sample's topography, morphology, and composition. The SEM provides valuable insights into the structure and characteristics of a wide range of materials, from biological specimens to metals, semiconductors, and geological samples.

    The FIB component of the Helios Dual-Beam microscope is where its true power lies. It utilizes a finely focused beam of ions, typically gallium ions, to remove material from the surface of a sample through a process known as milling. This milling capability allows for precise material removal, making it possible to create cross-sections, trenches, and three-dimensional structures with nanoscale precision. Additionally, the FIB can deposit materials, such as metals or insulators, onto specific areas of a sample, enabling localized modification or repair.

Helios Dual-Beam image of gold nanoparticle sample milling

    The Helios Dual-Beam microscope finds applications in various fields. In the semiconductor industry, it aids in failure analysis, defect localization, and circuit editing. It assists in modifying and repairing integrated circuits by accurately removing or depositing materials at the nanoscale level. In materials science, it provides insights into the microstructure, surface properties, and composition of advanced materials like metals, alloys, ceramics, and composites. Additionally, the FIB functionality enables the fabrication of precise samples for further analysis using techniques like transmission electron microscopy (TEM).

    The Helios Dual-Beam microscope is also essential in nanotechnology research and development, allowing for the creation of nanostructures, nanowires, and nanopatterns with exceptional precision. In life sciences, it aids in high-resolution imaging of cellular structures, tissues, and biomaterials. It enables detailed analysis and visualization of biological samples, including cross-sectioning for examining internal structures or generating 3D reconstructions of complex architectures.

    During our exploration, we observed the Helios Dual-Beam microscope in action. It demonstrated its laser cutting tool on a sample of gold containing nanoscale pores. The instrument showcased its capability to manipulate challenging samples, including alloys that are difficult to mix evenly. We also encountered a sample composed of many different metals analyzed using EDS to highlight the versatility of the microscope.

Example EDS display

    When working with biological samples, it is crucial to consider that materials transparent to the naked eye may not be transparent to electrons. Instead, electrons may bounce off the sample surface, limiting the level of detail obtained. Cross-sectioning is a technique employed to examine what lies beneath the surface of a sample. By employing microtomes within the SEM, it is possible to slice the sample and capture images, ultimately leading to the construction of a 3D image.  Another interesting technique demonstrated was the High-Resolution MicroCT of a gecko.

High-resolution Mirco-CT of a gecko

    At ASTeCC, we immersed ourselves in a community of curious minds united in their pursuit of scientific knowledge. The collaboration among scientists and researchers perfectly complemented the remarkable technologies we encountered. The Helios Dual-Beam microscope and other cutting-edge technologies unveiled boundless possibilities for exploration and innovation. Departing from ASTeCC, we carry newfound inspiration, ready to infuse the magic of nanoscale science into our own endeavors, just as Dorothy brought wisdom and courage back to Kansas. Our journey taught us that remarkable adventures and discoveries often lie just beyond the familiar, waiting to be unveiled with the right tools and the support of a passionate community. ASTeCC became our own Emerald City, where science and imagination converged, and the memories of this experience will forever inspire our scientific quests.

"Toto, I've got a feeling we're not in Kansas anymore."

- Dorothy Gale, The Wizard of Oz