diff --git a/00_introduction.md b/00_introduction.md index f858c4e..f3b784f 100644 --- a/00_introduction.md +++ b/00_introduction.md @@ -3,26 +3,26 @@ title: Introduction --- Sample preparation for scanning/transmission electron microscopy (S/TEM) is a critical and challenging aspect of materials characterization. -Conventional methods for producing the ultra-thin, <100 nm,samples typically required for S/TEM analysis [@https://doi.org/10.1007/978-1-4419-9244-4_5; @https://doi.org/10.1007/978-0-387-76501-3_10] are labor-intensive, often requiring hours to days of work by highly trained specialists. +Conventional methods for producing the ultra-thin, <100 nm samples typically required for S/TEM analysis [@https://doi.org/10.1007/978-1-4419-9244-4_5; @https://doi.org/10.1007/978-0-387-76501-3_10] are labor-intensive, often requiring hours to days of work by highly trained specialists. The ultra-thin sections must be representative of the bulk material while minimizing preparation-induced artifacts, such as contamination, and chemical and structural alteration. Sample thickness must be carefully controlled to suit both the sample heterogeneity and the types of analyses planned. -Excessive thickness obscures sample features, compromises both high-resolution image contrast and electron energy loss spectrum quality. +Excessive thickness obscures sample features, compromises both high-resolution image contrast and electron energy loss spectrum quality. Likewise, insufficient thickness compromises structural integrity and may result in the loss of critical microstructural information, as well as reduced image and spectral intensity. These challenges are compounded by the diverse range of materials studied and the growing demand for site-specific analyses. Continuous refinement of TEM sample preparation methods over more than 7 decades has greatly expanded the range of materials that can be analyzed effectively at ever more demanding requirements for spatial resolution and spectroscopic sensitivity. Electrolytic etching, mechanical polishing, broad-beam ion milling, mechanical grinding and drop casting, all have useful application histories [@blackham1993applications; @https://doi.org/10.1006/anbo.2001.1367; @https://doi.org/10.1007/978-1-4419-9244-4_5]. In recent years, the two most popular sample preparation methods are ultramicrotomy, particularly for soft materials, but also for mineralogical samples and fine-grained composites, and focused ion beam (FIB) milling for thin films and bulk materials, and anything site-specific. -In FIB milling cross-section preparation, a rastered beam of Ga+, or more recently a noble gas plasma, mills out a membrane of the target material. +In FIB milling cross-section preparation, a rastered beam of Ga+, or more recently a noble gas plasma, mills out a membrane of the target material. FIB preparation is now the default for most TEM studies of hard materials, due to the relative ease with which one can prepare uniform thickness, site-specific cross-sections of multiphase-materials. However, FIB instruments are an order of magnitude more expensive than microtomes, and significantly more wasteful of sample volume when preparing serial sections. -In ultramicrotomy sectioning, a sharp blade shaves a series of serial slices from a mounted sample. This method was first developed in the 1950s [@https://doi.org/10.3181/00379727-74-17931; @https://doi.org/10.1002/ar.1091170403; @https://doi.org/10.1002/jcp.1030420103] for sectioning biological specimens. +In ultramicrotomy sectioning, a sharp blade shaves a series of serial slices from a mounted sample. This method was first developed in the 1950s [@https://doi.org/10.3181/00379727-74-17931; @https://doi.org/10.1002/ar.1091170403; @https://doi.org/10.1002/jcp.1030420103] for sectioning biological specimens. Ultramicrotomy methods have improved significantly since the 1950s. A critical advancement was Humberto Fernández-Morán's development of the diamond knife in 1953-1955, which offered better sharpness and durability than glass knives and could section harder materials [@https://doi.org/bzp3r6]. Fernández-Morán also introduced cryo-ultramicrotomy in 1952, which preserved samples at low temperatures without chemical fixatives. -The microtomes themselves evolved from simple mechanical systems in the 1950s to modern, digitally-controlled systems with automated sectioning and greater precision and reproducibility [@reid1975practical; @https://doi.org/10.1007/978-1-4419-9244-4_5]. +The microtomes themselves evolved from simple mechanical systems in the 1950s to modern, digitally-controlled systems with automated sectioning and greater precision and reproducibility [@reid1975practical; @https://doi.org/10.1007/978-1-4419-9244-4_5]. Ultramicrotomy remains essential for ultrastructural investigations requiring high spatial resolution with minimal preparation-induced artifacts. Ultramicrotomy applications have expanded to include polymers, semiconductor cross-sectioning, nanoparticle distribution analysis, and coating examination. Geological and astromaterial applications include mineral composition and structure, organic-mineral composites, and radiation tracks, and coordinated studies on serial slices with different instruments. @@ -34,7 +34,7 @@ In order to use the ultramicrotome to section samples, the samples must be robus The optimal embedding material depends on the sample properties and which specific analyses are planned. Traditional embedding protocols employ epoxy resins that work well for conventional light optical studies, and transmission electron microscope studies under low beam current densities in moderate vacuum conditions. -Epoxy resins offer excellent sectioning properties and compatibility with standard staining protocols [@https://doi.org/10.1083/jcb.9.2.409] for low Z materials, and are also generally stable for storage for longitudinal studies. -However, epoxy resins can be problematic for polymer composite samples due to potential chemical contamination, thermal degradation during curing, and difficulty sectioning heterogeneous mineral assemblages [@https://doi.org/10.3390/life10080135; @plummer2025ultramicrotomy]. +Epoxy resins offer excellent sectioning properties and compatibility with standard staining protocols [@https://doi.org/10.1083/jcb.9.2.409] for low Z materials, and are also generally stable for storage for longitudinal studies. +However, epoxy resins can be problematic for polymer composite samples due to potential chemical contamination, thermal degradation during curing, and difficulty sectioning heterogeneous mineral assemblages [@https://doi.org/10.3390/life10080135; @plummer2025ultramicrotomy]. Epoxy resins are generally not robust to the probe current densities widely used in conventional field emission and aberration-corrected scanning transmission electron microscopes [@https://doi.org/10.1016/j.micron.2004.02.003; @https://doi.org/10.1017/S1431927608080902]. -Sulfur is a useful alternative to epoxy embedding because it provides the necessary mechanical robustness for the microtomy, and can be sublimated at low temperature after the section to leave the bare sample section. This eliminates the vacuum degradation from the epoxy outgassing in the electron microscope, sample contamination from volatilization and redeposition of the epoxy hydrocarbons and beam-induced shrinkage of the epoxy leading to sample mechanical instability in the microscope. This study presents a comprehensive sulfur-embedding protocol specifically optimized for preparing ultrathin sections (50-100 nm in thickness) particles with varied composition. We evaluate sectioning quality, sample preservation, and compatibility with advanced microscopic and spectroscopic techniques without conventional epoxy embedding methods. +Sulfur is a useful alternative to epoxy embedding because it provides the necessary mechanical robustness for the microtomy and can be sublimated at low temperature after sectioning to leave the bare sample section. This eliminates vacuum degradation from epoxy outgassing in the electron microscope, sample contamination from volatilization and redeposition of epoxy hydrocarbons, and beam-induced shrinkage of the epoxy leading to sample mechanical instability in the microscope. This study presents a comprehensive sulfur-embedding protocol specifically optimized for preparing ultrathin sections (50-100 nm in thickness) of particles with varied composition. We evaluate sectioning quality, sample preservation, and compatibility with advanced microscopic and spectroscopic techniques without conventional epoxy embedding methods. diff --git a/01_instrumentation.md b/01_instrumentation.md index 36a9fe6..2a0507d 100644 --- a/01_instrumentation.md +++ b/01_instrumentation.md @@ -3,7 +3,7 @@ title: Instrumentation --- ## Ultramicrotome -The essential components of a microtome are: a sample mounted in/on a bullet of epoxy; a chuck to secure the bullet; a knife blade; a mechanism to advance the sample towards the knife, and raise and lower the sample vertically at the knife edge. +The essential components of a microtome are: a sample mounted in or on a bullet of epoxy; a chuck to secure the bullet; a knife blade; a mechanism to advance the sample towards the knife, and raise and lower the sample vertically at the knife edge. Automated ultramicrotomes advance the specimen arm holding the embedded sample block towards the cutting knife edge with precision and reproducibility. The knife stage maintains the cutting edge at adjustable clearance angles, typically 6°, optimized for the specimen's properties. A stereomicroscope with appropriate illumination permits real-time monitoring of the sectioning process. The sample slices float off of the knife into a bath, usually filled with water. Either glass or diamond knives can be used, depending on the materials being cut and the desired thickness. @@ -27,7 +27,7 @@ The mechanical system controls the sectioning interval and determines section th ### Knife and Knife Holder: The ultramicrotome uses either a glass knife for initial rough cuts or diamond knife for most ultra-thin sectioning. -Diamond knives come in different angles, typically 35°or 45°, with edge lengths ranging from 1 to 7 mm depending on the application. +Diamond knives come in different angles, typically 35° or 45°, with edge lengths ranging from 1 to 7 mm depending on the application. The knife has an attached boat or trough that holds water, allowing the sections to float as they are cut. :::{figure} ./figures/fig_3.png @@ -75,4 +75,4 @@ The base platform holds and positions the sample holder, allowing for precise tr :::{figure} ./figures/fig_6b.png :name: stage_mounted **Custom removable stage for sulfur embedding.** A specialized stage was fabricated with increased mass for vibration dampening, thermal isolation to prevent external heat transfer, and precise leveling mechanisms. Isolation mounts decouple the working surface from building vibrations and mechanical disturbances, ensuring stable conditions during the critical sulfur solidification phase and precise specimen positioning. -::: \ No newline at end of file +::: diff --git a/02_methods.md b/02_methods.md index 094efd1..536eb5f 100644 --- a/02_methods.md +++ b/02_methods.md @@ -15,39 +15,57 @@ title: Methods The first step in preparing samples by ultramicrotomy is to mount the sample. In the case of sulfur embedding, the samples are placed in a droplet of molten sulfur, which solidifies around the sample particle, and then the particle encased in solidified sulfur is glued to the front of an epoxy bullet. It is useful to prepare a batch of epoxy bullets in advance, according to manufacturer specifications to create stable mounting substrates. The epoxy bullets provide standardized mounting blocks for the microtome specimens, ensuring secure attachment in the ultramicrotome chuck assembly and consistent alignment during sectioning, while eliminating time-consuming custom mounting procedures. These uniform cylindrical blocks, cast in silicone molds, offer superior specimen adhesion compared to temporary mounting methods and prevent sample detachment during sectioning of hard materials or serial section collection. Sulfur beads applied to the bullet's top surface serve as an intermediate mounting medium, providing enhanced adhesion for specific sample types while allowing easy specimen removal or re-orientation when necessary. -For ultramicrotomy of individual particles too small to move easily with tweezers, a micromanipulator is essential. For embedding high-value, one-of-kind samples, we use a fully computer controlled, dual- axis micromanipulator, e.g., the Microsupport Axis Pro micromanipulator, to position the particles in the sulfur.. This instrument allows users to manipulate particles with micrometer-level control in order to orient particles at specific depths within the sulfur matrix. Remote manipulation instead of direct handling can reduce the introduction of foreign materials into the sulfur matrix that might subsequently interfere with analytical measurements. -Several alternatives to Microsupport systems are available depending on application requirements and budget. Narishige hydraulic micromanipulators offer excellent manual control and stability for precision work at moderate cost. The ExLo lift-out system provides integrated ex-situ extraction capabilities for FIB-SEM environments. Manual particle picking using fine needles or precision tweezers under and optical microscope remains the most affordable and practical option, for particles > 10 𝝻m. The selection of the appropriate micromanipulator system depends on the required precision, sample characteristics, throughput needs, and available funding, with each approach offering distinct trade-offs between cost, functionality, and operational complexity. -The Axis Pro has a stable mechanical design that minimizes vibrations that could disrupt the delicate positioning, and the precise control enables consistency in the embedding protocol. To further enhance stability during the embedding process, we fabricated a custom removable stage (@https://ntrs.nasa.gov/citations/20250003593) (Plummer, 2024) to provide a more rigid and thermally stable platform than the standard microscope stage, incorporating features such as increased mass for vibration dampening, thermal isolation to prevent heat transfer from external sources, and precise leveling mechanisms to ensure optimal working conditions during the critical sulfur solidification phase. This specialized stage utilizes isolation mounts that effectively decouple the working surface from external mechanical disturbances, including building vibrations, footsteps, and equipment-generated oscillations that could disrupt precise positioning during sulfur embedding. + +For ultramicrotomy of individual particles too small to move easily with tweezers, a micromanipulator is essential. For embedding high-value, one-of-a-kind samples, we use a fully computer-controlled, dual-axis micromanipulator, e.g., the Microsupport Axis Pro micromanipulator, to position the particles in the sulfur. This instrument allows users to manipulate particles with micrometer-level control in order to orient particles at specific depths within the sulfur matrix. Remote manipulation instead of direct handling can reduce the introduction of foreign materials into the sulfur matrix that might subsequently interfere with analytical measurements. + +Several alternatives to Microsupport systems are available depending on application requirements and budget. Narishige hydraulic micromanipulators offer excellent manual control and stability for precision work at moderate cost. The ExLo lift-out system provides integrated ex-situ extraction capabilities for FIB-SEM environments. Manual particle picking using fine needles or precision tweezers under an optical microscope remains the most affordable and practical option, for particles > 10 𝝻m. The selection of the appropriate micromanipulator system depends on the required precision, sample characteristics, throughput needs, and available funding, with each approach offering distinct trade-offs between cost, functionality, and operational complexity. + +The Axis Pro has a stable mechanical design that minimizes vibrations that could disrupt the delicate positioning, and the precise control enables consistency in the embedding protocol. To further enhance stability during the embedding process, we fabricated a custom removable stage ([Plummer, 2024](https://ntrs.nasa.gov/citations/20250003593 "Plummer, 2024")) to provide a more rigid and thermally stable platform than the standard microscope stage, incorporating features such as increased mass for vibration dampening, thermal isolation to prevent heat transfer from external sources, and precise leveling mechanisms to ensure optimal working conditions during the critical sulfur solidification phase. This specialized stage utilizes isolation mounts that effectively decouple the working surface from external mechanical disturbances, including building vibrations, footsteps, and equipment-generated oscillations that could disrupt precise positioning during sulfur embedding. + To provide a thermally stable working surface for sulfur embedding procedures, we mounted a uniformly heated quartz plate (TC-HPQ75x50, 1.1 mm thickness) on the custom removable stage. The plate features a 75×50 mm heating surface with a 70×45 mm optical window and integrated temperature sensor for precise thermal control. Temperature regulation was maintained using a TC-1-100s-24V temperature controller, ensuring uniform heat distribution across the quartz surface during particle manipulation and embedding. The plate's 128×86 mm mounting frame provided stable integration with the removable stage while allowing optical access for specimen visualization and documentation throughout the embedding process. While the heated quartz plate and its efficacy are described in this manuscript, a hotplate and an optical microscope with reflected light observation, can be substituted. ## Particle Picking and Sulfur Embedding -Manipulation of particulate samples, a.k.a. particle picking, requires a systematic approach combining proper equipment, careful technique, and stringent contamination controls. The essential setup includes a stereo microscope with 10-50x magnification and/or micromanipulator with fine positioning controls, clean glass slides, clean picking tools such as borosilicate glass needles, and precise sample mounting stage with previously discussed specifications, all operated within a clean work environment. If using the heated quartz plate and removable stage, these need to be set up on the micromanipulator stage so that sample can be seen and documented with both a glass slide with elemental sulfur on it, and a separate glass slide with target material sample grains. Sample preparation begins with mounting particle samples, here we use powdered meteorite matrix on clean glass slides, and documenting target areas of particles thoroughly before picking. The heated plate is then gradually heated from ambient temperature to 90°C, then incrementally increased to 114-116°C, while monitoring the droplet sulfur melting under optical microscopy. Complete sulfur melting is confirmed by observing fluid behavior and surface tension characteristics. Heat sensitive samples should be kept on an unheated glass slide, until ready to transfer into the molten sulfur to minimize heating time and temperature. -The particle picking technique involves first locating target particles under low magnification, before switching to higher magnification for precise particle work. Once particles are identified and documented, individual locations can be saved, in case of a programmable stage, and appropriately calibrated. Next, the location for the sulfur droplets on the heated quartz plate needs to be identified and set as a saved stage position. Approach particles by bringing the glass needle slowly into focus down towards the stage using the finest control settings to avoid overshooting or damage. Contact should be made gently, maintaining consistent magnification and using steady, controlled movements to transfer particles into molten sulfur medium. -Individual particles can be transferred into molten sulfur droplets and positioned below the surface using electrostatic positioning techniques. The arm-attachment functions of the computer-controlled micromanipulator allow the microscopist to move particles with a glass or tungsten needle to the droplet, and laterally within the sulfur droplet or deeper below the surface to achieve optimal embedding depth. Particle placement should be optimized to ensure uniform sulfur distribution around the sample while minimizing air bubble incorporation. The crystallization of sulfur over the sample is critical for maintaining precise particle positioning achieved during electrostatic manipulation and creating a mechanically stable matrix that prevents specimen movement during sectioning. Complete crystallization ensures uniform cutting properties across the block face, eliminating voids or soft regions that could cause sectioning artifacts such as knife chatter, compression, or specimen pullout during microtomy. The solid crystalline structure provides the mechanical integrity necessary for ultrathin sectioning while preserving the particle's orientation relative to the cutting plane, which is essential for exposing targeted internal features or interfaces established during the initial positioning process. +Manipulation of particulate samples, a.k.a. particle picking, requires a systematic approach combining proper equipment, careful technique, and stringent contamination controls. The essential setup includes a stereo microscope with 10-50x magnification and/or micromanipulator with fine positioning controls, clean glass slides, clean picking tools such as borosilicate glass needles, and precise sample mounting stage with previously discussed specifications, all operated within a clean work environment. If using the heated quartz plate and removable stage, these need to be set up on the micromanipulator stage so that sample can be seen and documented with both a glass slide with elemental sulfur on it, and a separate glass slide with target material sample grains. Sample preparation begins with mounting particle samples, here we use powdered meteorite matrix on clean glass slides, and documenting target areas of particles thoroughly before picking. The heated plate is then gradually heated from ambient temperature to 90°C, then incrementally increased to 114-116°C, while monitoring the droplet sulfur melting under optical microscopy. Complete sulfur melting is confirmed by observing fluid behavior and surface tension characteristics. Heat-sensitive samples should be kept on an unheated glass slide until ready to transfer into the molten sulfur to minimize heating time and temperature. + +The particle picking technique involves first locating target particles under low magnification, before switching to higher magnification for precise particle work. Once particles are identified and documented, individual locations can be saved, in the case of a programmable stage, and appropriately calibrated. Next, the location for the sulfur droplets on the heated quartz plate needs to be identified and set as a saved stage position. Approach particles by bringing the glass needle slowly into focus down towards the stage using the finest control settings to avoid overshooting or damage. Contact should be made gently, maintaining consistent magnification and using steady, controlled movements to transfer particles into molten sulfur medium. + +Individual particles can be transferred into molten sulfur droplets and positioned below the surface using electrostatic positioning techniques. The arm-attachment functions of the computer-controlled micromanipulator allow the microscopist to move particles with a glass or tungsten needle to the droplet, and laterally within the sulfur droplet or deeper below the surface to achieve optimal embedding depth. Particle placement should be optimized to ensure uniform sulfur distribution around the sample while minimizing air bubble incorporation. The crystallization of sulfur over the sample is critical for maintaining precise particle positioning achieved during electrostatic manipulation and creating a mechanically stable matrix that prevents specimen movement during sectioning. Complete crystallization ensures uniform cutting properties across the block face, eliminating voids or soft regions that could cause sectioning artifacts such as knife chatter, compression, or specimen pullout during microtomy. The solid crystalline structure provides the mechanical integrity necessary for ultrathin sectioning while preserving the particle's orientation relative to the cutting plane, which is essential for exposing targeted internal features or interfaces established during the initial positioning process. + The central placement of particles within sulfur droplets is critical to ensure uniform embedding matrix thickness on all sides of the specimen, preventing asymmetric support that could lead to differential cutting forces and sectioning artifacts during microtomy. When particles are positioned off-center, thin sulfur regions on one side may cause specimen pullout or chipping, while excessively thick regions create uneven resistance that results in knife chatter or section compression. Central positioning also maximizes the number of useful sections that can be obtained from the embedded block, as it ensures the particle remains within the sectioning plane for the greatest possible depth, providing more opportunities to examine different levels of the specimen's internal structure. Additionally, centrally embedded particles experience more uniform thermal stress during temperature changes, reducing the likelihood of matrix cracking or specimen displacement that could compromise the integrity of the embedded sample during storage or sectioning procedures. + Controlled sulfur cooling with the introduction of nucleation points ensures uniform crystallization that prevents specimen displacement and promotes optimal sectioning properties. Nucleation points can be introduced through mechanical agitation or contact with a cool probe to initiate controlled crystallization from multiple sites throughout the sulfur matrix, promoting fine-grained crystal structure that provides uniform mechanical properties around the embedded particle. Without controlled nucleation, sulfur tends to form large, irregular crystals that create internal stress concentrations and preferential cleavage planes that compromise sectioning quality. The timing of nucleation point introduction requires careful consideration - initiating crystallization too early while sulfur remains highly fluid can disrupt particle positioning, while delayed nucleation may result in uncontrolled crystal growth that creates voids or inclusions around the specimen. Gradual cooling combined with strategic nucleation point placement ensures that crystallization progresses uniformly inward from multiple locations, minimizing thermal gradients that could cause differential contraction and specimen movement during the solidification process, ultimately producing a homogeneous crystalline matrix suitable for consistent ultrathin sectioning. + Quality control measures include taking before and after photographs of picking areas, maintaining detailed records of particle locations and characteristics and verifying particle identity under higher magnification in the sulfur droplet before final collection, with success ultimately depending on patience, steady technique, and maintaining contamination-free conditions throughout the entire process. ### Knife Preparation and Setup -Glass knives are fabricated by scoring and fracturing specialized glass strips, producing fresh cutting edges for each use. We prepare glass knives with a Leica EM KMR3 glass knifemaker. A knife thickness of 6.4 mm enables optimal cutting performance for ultrathin sections for electron microscopy. While economical and suitable for routine applications, glass knives exhibit limited durability. Edge degradation occurs rapidly with use, necessitating frequent replacement. Glass knives are employed for rough cutting and preliminary microtome sectioning as a cost-effective alternative to diamond knife trimming tools during the initial stages of this sample preparation protocol. While diamond knives are reserved for final ultrathin sectioning (50-100 nm), glass knives are ideal for block trimming, semi-thin sectioning (0.5-2 μm), and survey sections due to their disposable nature and adequate cutting performance. This approach allows for aggressive cutting without risk of damaging expensive precision instruments, enabling researchers to remove excess resin, shape block faces, and evaluate sample quality through light microscopy before proceeding to final ultrathin sectioning. Although glass knives may produce more compression artifacts and chatter marks compared to diamond knives, particularly with harder samples, their fixed cutting geometry (typically 45°) provides sufficient quality for preparatory work while maximizing the lifespan of diamond knives for critical high-resolution applications. +Glass knives are fabricated by scoring and fracturing specialized glass strips, producing fresh cutting edges for each use. We prepare glass knives with a Leica EM KMR3 glass knifemaker. A knife thickness of 6.4 mm enables optimal cutting performance for ultrathin sections for electron microscopy. While economical and suitable for routine applications, glass knives exhibit limited durability. Edge degradation occurs rapidly with use, necessitating frequent replacement. Glass knives are employed for rough cutting and preliminary microtome sectioning as a cost-effective alternative to diamond knife trimming tools during the initial stages of this sample preparation protocol. While diamond knives are reserved for final ultrathin sectioning (50-100 nm), glass knives are ideal for block trimming, semi-thin sectioning (0.5-2 μm), and survey sections due to their disposable nature and adequate cutting performance. This approach allows for aggressive cutting without risk of damaging expensive precision instruments, enabling researchers to remove excess resin, shape block faces, and evaluate sample quality through light microscopy before proceeding to final ultrathin sectioning. Although glass knives may produce more compression artifacts and chatter marks compared to diamond knives, particularly with harder samples, their fixed cutting geometry (typically 45°) provides sufficient quality for preparatory work while maximizing the lifespan of diamond knives for critical high-resolution applications. + Diamond knives feature precision-cut diamond edges and are ideal for demanding applications and final sectioning. These knives maintain cutting quality for extended periods (months to years with proper maintenance) and can produce consistently thinner sections down to 20 nm. Diamond knives are manufactured in various geometries (25°, 35°, 45° edge angles) to accommodate different material properties. As the knife edge angle decreases, such as 35°, the produced sections have reduced compression of sections, and works well for hard, well-infiltrated specimens. Alternatively, a 45° knife edge angle is more durable, making it less prone to chipping. While the edge dulls less quickly, it may cause more compression during sectioning. This angle is better suited for harder materials or semi-thin sections and is common for glass knives, which are more fragile than diamond knives. + Despite substantial initial investment, the longevity of diamond knives renders them cost-effective for high-volume work. Each knife is fitted with a boat—a small reservoir that retains water. Sections float onto this liquid surface immediately upon cutting, where surface tension prevents collapse and maintains section flatness. ## Ultramicrotomy: Sectioning Procedure, Section Cutting, and Collection In ultramicrotomy, initial rough cuts create a trapezoidal-shaped block face (commonly called a pyramid) as a crucial preparatory step before cutting thin sections. The pyramid, typically measuring 0.5-1.0 mm on each side, trims the embedded specimen to a small, precisely shaped face containing only the sample of interest. This reduced cutting face minimizes extraneous forces during sectioning, reduces section compression, and supports sulfur stability. Rough cutting can be accomplished through manual trimming with fresh razor blades or mechanically operated trimming using the ultramicrotome equipped with a glass knife. The trimming process begins with examining the embedded block under a stereomicroscope to locate the sample. Excess material is then removed from all sides, working progressively closer to the target tissue while maintaining a trapezoidal geometry. The top face (the future cutting face) should be slightly smaller than the bottom, creating a trapezoid when viewed from the side. As trimming progresses closer to the tissue, finer, more controlled cuts are made to avoid specimen damage or sulfur instability. + The trapezoidal block face shape serves several functions: the parallel top and bottom edges allow sections to form straight, aligned ribbons for easier collection; the shorter top edge experiences less resistance than a square face, reducing chatter and compression; and the slight taper prevents the knife from contacting the block sides during sectioning. For electron microscopy, the final trimmed face should be sufficiently small to minimize section area and maximize image quality. + The trimmed block is secured in the ultramicrotome chuck and positioned relative to the knife edge. Alignment is critical and is typically achieved by observing the knife-edge reflection on the block face. Cutting parameters are established, including section thickness, cutting speed, and knife clearance angle. + Sectioning was performed using a Leica EM UC6 ultramicrotome equipped with a 45° diamond knife (DIATOME Ultra 45) and ultrapure water boat. Sample blocks were initially trimmed using a fresh glass knife chisel to create trapezoidal faces with optimal cutting geometry. During the cutting stroke, the specimen arm advances the block toward the knife in an arc. The knife edge shears a thin section from the block face, which immediately floats onto the water surface in the knife boat. Multiple consecutive sections often remain attached, forming ribbons. Trimming continued until embedded particles were positioned 1-2 μm below the cutting surface. Sections were prepared at cutting speeds of 0.5-1.0 mm/s with feed increments of 50-70 nm. Sequential sections were collected as continuous ribbons on the water surface and evaluated for quality based on uniform thickness, absence of knife marks, and minimal compression artifacts. + Ultrathin sections are collected by floating them onto the surface of a water-filled boat attached to the diamond knife. As sections are cut, they drift away from the knife edge and float on the water surface, often forming ribbons of consecutive sections. Section ribbons are retrieved from the water surface using fine tools such as eyelash probes or wire loops and collected onto support grids. + An eyelash tool is a single hair glued to a wooden stick or applicator used to gently manipulate, position, and separate ultrathin sections floating on the water surface in the knife boat during electron microscopy sample preparation, taking advantage of the eyelash's natural fineness and flexibility to handle delicate sections without causing damage. A perfect loop is a simple wire tool consisting of a thin wire formed into a small circular loop at the end of a handle. The loop diameter is usually 2-4mm, roughly matching or slightly larger than a 3 mm TEM grid that works by exploiting water's surface tension to create a stable water film across the loop opening. + Standard TEM grids are 3 mm in diameter, constructed of copper, nickel, or gold, with various mesh configurations (100-400 mesh). To transfer sections to TEM grids, a grid is grasped with fine-tipped forceps and carefully positioned beneath the floating sections, then lifted vertically to capture them on its surface. Alternatively, the grid can be approached from above and gently touched to the section before lifting. Excess water is wicked away by touching the edge of the grid to filter paper, and the grid is allowed to dry completely before examination. The sections adhere to the grid through surface tension and van der Waals forces, with the carbon support film (if present) providing additional stability. ### Support Grids Copper is the most common grid material for routine transmission electron microscopy applications. Copper grids are widely used because they are inexpensive and work well for most materials with different sizes. The material provides good mechanical strength and electrical conductivity, which helps prevent charge buildup under the electron beam. However, copper can react with acidic solutions used in specimen preparation and may oxidize over time. + For routine TEM use, copper grids covered with carbon film work well in many cases and provide excellent results. The choice of grid material depends on the specimen type, chemical compatibility with preparation procedures, temperature requirements, magnetic properties of the sample, analytical needs such as EDS, and cost. Specialized applications requiring chemical resistance, high temperatures, or freedom from magnetic interference may require alternative materials despite their higher cost. -#### Artifact Management -Several artifacts may compromise microtome section quality (Reis, 1975; Hayat, 2000; Dykstra and Reuss, 2003). Chatter refers to a characteristic defect pattern caused by the vibration of the knife or knife-edge that can result in the blade alternately slipping and sticking on the sample, which leaves a characteristic lateral stripes from variations in the sample slice thickness. This defect can be minimized by ensuring that the ultramicrotome is situated on a table with vibration-isolation, and that the sample is fully enclosed in the embedding media, so that the knife has adequate physical support to avoid slip. Accordion-like distortions that compress the sample sections due to resistance during cutting and may be minimized by reducing cutting speed or knife angle. Knife marks appear as linear scratches caused by debris on the knife edge or edge damage. Thickness variations result from mechanical instability or thermal fluctuations in the advancement mechanism. Section loss occurs when sections sink or fold, typically due to electrostatic effects or improper water levels. +### Artifact Management +Several artifacts may compromise microtome section quality (Reid, 1975; Hayat, 2000; Dykstra and Reuss, 2003). Chatter refers to a characteristic defect pattern caused by the vibration of the knife or knife-edge that can result in the blade alternately slipping and sticking on the sample, which leaves characteristic lateral stripes from variations in the sample slice thickness. This defect can be minimized by ensuring that the ultramicrotome is situated on a table with vibration isolation, and that the sample is fully enclosed in the embedding media, so that the knife has adequate physical support to avoid slip. Accordion-like distortions that compress the sample sections due to resistance during cutting may be minimized by reducing cutting speed or knife angle. Knife marks appear as linear scratches caused by debris on the knife edge or edge damage. Thickness variations result from mechanical instability or thermal fluctuations in the advancement mechanism. Section loss occurs when sections sink or fold, typically due to electrostatic effects or improper water levels. diff --git a/03_steps.md b/03_steps.md index c12a850..8d998f0 100644 --- a/03_steps.md +++ b/03_steps.md @@ -9,7 +9,7 @@ title: Particle Picking and Sulfur Embedding Protocol - Stage Calibration: Calibrate precise sample mounting stage using reference standards. Verify repeatability of positioning to within ±1 μm. Test stage stability under vibration and thermal conditions. Ensure smooth operation of all mechanical adjustments without drift or play. :::{figure} ./figures/fig_5.png -:name: fig_5 +:name: micromanipulator_setup **Micromanipulator for precision specimen manipulation.** The Microsupport Axis Pro micromanipulator provides multi-axis control for precise positioning and manipulation of specimens during microscopy and sample preparation. The device features independent X, Y, and Z-axis adjustment with fine-scale control, allowing operators to achieve micrometer-level positioning accuracy. ::: @@ -22,11 +22,11 @@ title: Particle Picking and Sulfur Embedding Protocol - Optical Integration: Position heated plate system to maintain optimal working distance for microscope objectives. Verify that heating elements do not interfere with illumination or create optical artifacts. Test visibility and focus quality at all required magnifications during heating cycles. :::{figure} ./figures/fig_6a.png -:name: removable_stage +:name: removable_stage_setup ::: :::{figure} ./figures/fig_6b.png -:name: stage_mounted +:name: stage_mounted_setup **Custom removable stage for sulfur embedding.** A specialized stage was fabricated with increased mass for vibration dampening, thermal isolation to prevent external heat transfer, and precise leveling mechanisms. Isolation mounts decouple the working surface from building vibrations and mechanical disturbances, ensuring stable conditions during the critical sulfur solidification phase and precise specimen positioning. ::: @@ -70,7 +70,7 @@ Sample Preparation and Documentation :::{figure} ./figures/particle_picking.png :name: particle_picking_micromanipulator -**Micrograph of target particles and glass needles for transferrring to molten sulfur.** +**Micrograph of target particles and glass needles for transferring to molten sulfur.** ::: ### System Calibration @@ -224,5 +224,3 @@ Comprehensive Photography Protocol: - Sample Labeling Protocol: Apply comprehensive labeling system including unique sample identification numbers, preparation date, operator identification, and critical preparation parameters. Use permanent marking methods resistant to solvents and mechanical handling during subsequent processing. - Quality Pre-Assessment: Conduct final quality assessment before releasing samples for sectioning including visual inspection, dimensional verification, and mechanical integrity testing. Document sample condition and any recommendations or precautions for sectioning personnel. - - diff --git a/04_steps_microtomy.md b/04_steps_microtomy.md index a40b5e2..edfb5ac 100644 --- a/04_steps_microtomy.md +++ b/04_steps_microtomy.md @@ -16,7 +16,7 @@ title: Microtomy Protocol ::: :::{figure} ./figures/fig_4.png -:name: sample_chuck +:name: sample_chuck_mounting **Mounting the specimen chuck into the ultramicrotome specimen arm.** The specimen chuck, containing the embedded sample block, is securely inserted into the specimen arm holder of the ultramicrotome. Proper mounting ensures stable specimen positioning and prevents vibration during sectioning. The chuck is aligned and tightened to maintain the correct orientation of the sample block relative to the knife edge, with the block face positioned perpendicular to the cutting direction for optimal sectioning quality. ::: @@ -94,11 +94,11 @@ Move the knife back several steps (25 μm) to avoid accidentally crashing the su **Final cut face of the sulfur sample after polishing with the glass knife.** Note the bright, mirror-like reflections from the fully polished edges, indicating excellent surface quality and readiness for diamond knife sectioning. ::: -Shavings should look "fronthy" and evolve into a translucent, solid peel. +Shavings should look "frothy" and evolve into a translucent, solid peel. :::{figure} ./figures/final_rough_cutting.mp4 :name: rough_cut_final -**Final stage of rough cutting and polishing the pyrimid face.** Video illustrating the final steps of surface polishing of the block face using a glass knife to achieve optical flatness before transition to a diamond knife, ensuring that residual chatter marks, compression artifacts, and surface irregularities introduced during rough cutting are minimized. +**Final stage of rough cutting and polishing the pyramid face.** Video illustrating the final steps of surface polishing of the block face using a glass knife to achieve optical flatness before transition to a diamond knife, ensuring that residual chatter marks, compression artifacts, and surface irregularities introduced during rough cutting are minimized. ::: :::{figure} ./figures/fig_23.png @@ -147,7 +147,7 @@ Shavings should look "fronthy" and evolve into a translucent, solid peel. :::{tip} :class: myclass1 myclass2 -:name: a-tip-reference +:name: section_integrity_tip Continuously check for section integrity such as: - Compression artifacts - Tears, folds, or separations @@ -178,7 +178,7 @@ Continuously check for section integrity such as: ::: :::{figure} ./figures/Fig2_mm.jpg -:name: ideal_ribbon +:name: ideal_ribbon_still **High-quality ribbon of consecutive 50 nm sulfur sections in the water bath.** Note the excellent uniformity in section shape and thickness throughout the ribbon, demonstrating ideal ultramicrotomy conditions suitable for high-resolution TEM analysis. Color has transitioned from highly reflective blue and gold to nearly transparent. Section A is the first sample slice, and Section B is the final section cut (shown in ribbon on left). They are shown side by side (right) to demonstrate what the goal is for a final section. This figure demonstrates common features: chatter (green), sample plucking (teal), and compression defects in the sample (magenta), section buckling and ripping (white dashed lines) in the sulfur section matrix. Section B demonstrates a section in the same ribbon but further in the process, with minimal chatter, less sample plucking, less compression defect, minimal ripping, no wrinkles in sulfur section matrix. ::: @@ -201,7 +201,7 @@ Continuously check for section integrity such as: :::{tip} :class: myclass1 myclass2 -:name: a-tip-reference +:name: floating_sections_tip Accidentally touching the middle of the floating sections can destroy sections altogether. Avoid this by working slowly and touching only near the edges and "paddling" the water around the sections to move them. ::: @@ -220,7 +220,7 @@ The operator grasps a TEM grid by its edge using fine-tipped tweezers. The grid :::{tip} :class: myclass1 myclass2 -:name: a-tip-reference +:name: grid_tweezer_tip Never grip grids with tweezers into film, only clip on the metal frame or the film/samples will be damaged. ::: @@ -237,7 +237,7 @@ Never grip grids with tweezers into film, only clip on the metal frame or the fi **View of the mounted sulfur ribbon showing the actual sample (in magenta) embedded within the sections, allowing for assessment of sample preservation and distribution within the ultrathin sections.** ::: -#### Critical Success Factors +### Critical Success Factors - Maintain minimal contamination conditions at all times. - Exercise patience during all manipulation procedures. - Use steady, controlled techniques for all movements. @@ -245,10 +245,7 @@ Never grip grids with tweezers into film, only clip on the metal frame or the fi :::{tip} :class: myclass1 myclass2 -:name: a-tip-reference -**Documentation is extremley important before baking and after.** Document every step thoroughly for reproducibility and troubleshooting. The sulfur sections are easy to see in reflected light, but identifying the locations of the sample in the sulfur will ultimately help identify sample locations post-sublimation. +:name: documentation_tip +**Documentation is extremely important before baking and after.** Document every step thoroughly for reproducibility and troubleshooting. The sulfur sections are easy to see in reflected light, but identifying the locations of the sample in the sulfur will ultimately help identify sample locations post-sublimation. ::: - - - diff --git a/06_conclusion.md b/05_results.md.md similarity index 58% rename from 06_conclusion.md rename to 05_results.md.md index 305172a..247dcae 100644 --- a/06_conclusion.md +++ b/05_results.md.md @@ -1,16 +1,16 @@ --- -title: Results and Conclusions +title: Results --- ## Results -:::{figure} ./figures/Fig4_mm +:::{figure} ./figures/Fig4_mm.jpg :name: final_example_tem_sample Dark field STEM image of a ~ 50 nm microtomed section of a macromolecular polymer after sulfur sublimation. The porous polymer section is suspended over a hole in a carbon film, suitable for background-free nanoscale imaging and spectroscopy. ::: Ultramicrotomy of sulfur-embedded samples yielded high-quality ribbons with minimal sectioning artifacts. The optimal particle size range for this protocol was determined to be 10-15 μm diameter, with larger particles (>15 μm) exhibiting increased susceptibility to plucking artifacts after 10-15 successful sections. Particle plucking occurred when mechanical stress concentrated at the boundaries between particles and the surrounding matrix material. This problem was most severe in areas containing hard mineral particles that had very different stiffness properties compared to the matrix. The plucking happened when the cutting process reached the widest part of each particle, indicating that the bond between the particle and matrix wasn't strong enough to withstand the cutting forces at that point. Quality assessment revealed that a majority of sections met criteria for TEM analysis (uniform thickness, minimal compression, minimal chatter), with 20-25 acceptable sections obtainable per particle before plucking onset. -:::{figure} ./figures/fig2_mm.jpg +:::{figure} ./figures/Fig2_mm.jpg :name: section_ribbon **Optical images of sulfur sections floating in the microtome boat.** Section A shows the first sample slice and Section B the final section cut, presented side by side to illustrate the goal for a final section. Common sectioning artifacts are annotated: chatter (green), sample plucking (teal), compression defects (magenta), and section buckling and ripping (white dashed lines) in the sulfur section matrix. Section B, taken from the same ribbon but later in the cutting process, demonstrates marked improvement with minimal chatter, reduced sample plucking and compression defects, minimal ripping, and no wrinkling in the sulfur section matrix. (inset) High-quality ribbon of consecutive 50 nm sulfur sections in the water bath, showing excellent uniformity in section shape and thickness throughout the ribbon — indicative of ideal ultramicrotomy conditions suitable for high-resolution TEM analysis. ::: @@ -36,28 +36,3 @@ Ultramicrotomy of sulfur-embedded samples yielded high-quality ribbons with mini :name: lacey_carbon_stem **TEM micrograph of sulfur-embedded specimen section on lacey carbon showing minimal sectioning artifacts.** Ultrathin section (~50 nm) collected using the sulfur droplet embedding method and deposited on lacey carbon grid displays minimal microtomy artifacts, with only minor chatter (periodic thickness variations creating faint parallel bands visible as sample loss over the lacey carbon holes). ::: - -## Conclusions - -Despite continuous advances in specimen preparation technology, ultramicrotomy remains irreplaceable for numerous applications requiring thin sections with minimal preparation-induced damage. The technique's combination of high spatial resolution, preservation of specimen architecture, and ability to generate multiple serial sections from a single specimen ensures its continued relevance in contemporary electron microscopy laboratories. Successful ultramicrotomy requires meticulous control of numerous variables, including cutting speed, knife angle, specimen hardness, air circulation, and water level. Even external factors, such as building vibrations, can influence results, necessitating the isolation of the ultramicrotome from mechanical disturbances. -The sulfur-embedding protocol presented here provides a viable and advantageous alternative to conventional epoxy methods for preparing ultrathin sections of both hard and soft materials. The technique offers superior sample retention, enhanced sectioning quality, and excellent preservation of original chemical compositions, making it particularly suitable for precious samples. Key protocol innovations include detailed procedures, precision particle positioning techniques, and systematic quality control metrics. The method's compatibility with high-resolution microscopic and spectroscopic techniques enables comprehensive characterization while minimizing sample consumption. -Future developments focus on extending the application of these procedures, optimizing embedding procedures for specific mineral assemblages and evaluating long-term sample stability under various storage conditions. This protocol contributes significantly to the expanding toolkit for non-destructive analysis of hard and soft material science research. As analytical capabilities continue to advance, ultramicrotomy will remain a cornerstone technique for ultrastructural investigation across diverse scientific disciplines. - - - -### Protocol Advantages and Limitations - -**Advantages:** -- Reduced contamination risk through elimination of organic solvents and accelerators -- Enhanced mechanical stability during sectioning due to improved particle-matrix adhesion -- Preservation of volatile and thermally sensitive phases through low-temperature processing -- Compatibility with multiple analytical techniques (TEM, STEM, SIMS) -- Improved handling characteristics and reduced sample loss during preparation - - -**Limitations:** -- Particle size restrictions based on sample hardness -- Potential for plucking artifacts in samples with high mineral hardness contrast -- Requirement for specialized temperature control equipment -- Limited long-term storage stability compared to epoxy embedding -- Issues with sulfur reactivity post-sublimation (copper sulfate dendrite) diff --git a/05_discussion.md b/06_discussion.md similarity index 92% rename from 05_discussion.md rename to 06_discussion.md index a0d9750..bcbb4e4 100644 --- a/05_discussion.md +++ b/06_discussion.md @@ -6,12 +6,12 @@ title: Discussion Epoxy resins have been the embedding medium of choice for ultramicrotomy for most samples since their introduction in the mid-1950s, due to their excellent ultrastructural preservation, minimal shrinkage, and reasonable mechanical stability under electron beam exposure [@10.1002/jemt.1070260604]. However, these resins undergo complex polymerization reactions involving cross-linking between epoxide groups, creating three-dimensional polymer networks of great mechanical strength with volumetric shrinkage typically between 2-7% during curing. Despite their widespread use, epoxy resins present several limitations for specialized applications. The polymerization process generates exothermic heat and involves chemical cross-linking that can potentially alter specimen chemistry and create artifacts through differential shrinkage. Additionally, epoxy resins do not penetrate tissues as effectively as less viscous formulations and may introduce unwanted chemical interactions in analytical applications requiring pristine chemical environments. These limitations are particularly problematic for precious samples from asteroid sample-return missions, where material conservation and analytical fidelity are paramount. -Sulfur-based embedding represents a specialized alternative that addresses these limitations through fundamentally different physical properties. A pioneer in using this method in the planetary science community was Bradley in his 2003 interplanetary dust particle analysis (@19940007627; @10.1016/B0-08-043751-6/01152-X). +Sulfur-based embedding represents a specialized alternative that addresses these limitations through fundamentally different physical properties. A pioneer in using this method in the planetary science community was Bradley in his 2003 interplanetary dust particle analysis (@bradley_carbon_1993; @10.1016/B0-08-043751-6/01152-X). The procedure was originally devised to measure energy-loss near-edge structure (ELNES) of light elements such as carbon, oxygen and nitrogen in organic material and enables X-ray absorption near-edge spectroscopy without contamination from the embedding medium [@10.1016/j.jaerosci.2016.02.008]. Previous studies have demonstrated the feasibility of sulfur embedding for biological specimens, but systematic protocols for hard and composite materials remain underdeveloped. Sulfur is a viable embedding medium because it undergoes a simple physical phase transition from liquid to solid at moderate temperature, without chemically reacting with most samples, and can be sublimated in air to leave embedding-media free sections. Sulfur-based embedding offers several potential advantages over conventional methods: chemical inertness toward most mineral phases, low processing temperatures, rapid solidification kinetics, minimal volume shrinkage during cooling, and improved stability in aberration corrected transmission electron microscopy. Sulfur has a high vapor pressure at room temperature; the ultra-thin sections are then placed in a separate vacuum chamber to sublimate the sulfur, yielding TEM specimens free of any embedding medium. This complete medium removal is a significant advantage, as it eliminates chemical interference during elemental analysis. Unlike epoxy resins that remain in sections and can interfere with spectroscopic analyses, sulfur embedding provides clean specimens ideal for studying organic materials and light elements where contamination-free analysis is critical, though it remains more specialized than epoxy for routine morphological studies. -This removability property has made sulfur embedding particularly valuable for energy-loss near-edge structure (ELNES) analysis of light elements and X-ray absorption near-edge spectroscopy (STXM-XANES) of extraterrestrial materials, where contamination from embedding media would compromise analytical results [@10.1016/j.gca.2003.10.044; @10.1016/j.gca.2010.05.010; @10.1126/science.1207758; @10.1111/j.1945-5100.2011.01237.x; @10.1186/s40623-014-0156-0; @10.1038/s41598-019-39357-1]. The technique has been applied to the analysis of samples, where preservation of original carbon, oxygen, and nitrogen chemical states is crucial for preservation of original materials. Furthermore, the absence of polymerization-induced shrinkage in sulfur embedding provides superior dimensional accuracy compared to epoxy systems, making it advantageous for applications requiring precise morphological measurements or when dealing with thermally sensitive specimens that cannot tolerate the exothermic curing conditions of epoxy resins. +This removability property has made sulfur embedding particularly valuable for energy-loss near-edge structure (ELNES) analysis of light elements and X-ray absorption near-edge spectroscopy (STXM-XANES) of extraterrestrial materials, where contamination from embedding media would compromise analytical results [@10.1016/j.gca.2003.10.044; @10.1016/j.gca.2010.05.010; @10.1126/science.1207758; @10.1111/j.1945-5100.2011.01237.x; @10.1186/s40623-014-0156-0; @10.1038/s41598-019-39357-1]. The technique has been applied to the analysis of samples where preservation of original carbon, oxygen, and nitrogen chemical states is crucial for preserving original materials. Furthermore, the absence of polymerization-induced shrinkage in sulfur embedding provides superior dimensional accuracy compared to epoxy systems, making it advantageous for applications requiring precise morphological measurements or when dealing with thermally sensitive specimens that cannot tolerate the exothermic curing conditions of epoxy resins. ### Protocol Advantages - Reduced contamination risk through elimination of organic solvents and accelerators @@ -23,7 +23,7 @@ This removability property has made sulfur embedding particularly valuable for e ### Protocol Limitations: - Particle size restrictions based on sample hardness - Potential for plucking artifacts in samples with high mineral hardness contrast -Requirement for specialized temperature control equipment +- Requirement for specialized temperature control equipment - Limited long-term storage stability compared to epoxy embedding - Issues with sulfur reactivity post-sublimation (copper sulfate dendrite) @@ -71,5 +71,3 @@ Quantifoil is a perforated support foil with precisely pre-defined hole size, sh - - diff --git a/07_conclusion.md b/07_conclusion.md new file mode 100644 index 0000000..631f315 --- /dev/null +++ b/07_conclusion.md @@ -0,0 +1,27 @@ +--- +title: Conclusions +--- +## Summary and Outlook + +Despite continuous advances in specimen preparation technology, ultramicrotomy remains irreplaceable for numerous applications requiring thin sections with minimal preparation-induced damage. The technique's combination of high spatial resolution, preservation of specimen architecture, and ability to generate multiple serial sections from a single specimen ensures its continued relevance in contemporary electron microscopy laboratories. Successful ultramicrotomy requires meticulous control of numerous variables, including cutting speed, knife angle, specimen hardness, air circulation, and water level. Even external factors, such as building vibrations, can influence results, necessitating the isolation of the ultramicrotome from mechanical disturbances. +The sulfur-embedding protocol presented here provides a viable and advantageous alternative to conventional epoxy methods for preparing ultrathin sections of both hard and soft materials. The technique offers superior sample retention, enhanced sectioning quality, and excellent preservation of original chemical compositions, making it particularly suitable for precious samples. Key protocol innovations include detailed procedures, precision particle positioning techniques, and systematic quality control metrics. The method's compatibility with high-resolution microscopic and spectroscopic techniques enables comprehensive characterization while minimizing sample consumption. +Future developments focus on extending the application of these procedures, optimizing embedding procedures for specific mineral assemblages and evaluating long-term sample stability under various storage conditions. This protocol contributes significantly to the expanding toolkit for non-destructive analysis of hard and soft material science research. As analytical capabilities continue to advance, ultramicrotomy will remain a cornerstone technique for ultrastructural investigation across diverse scientific disciplines. + + + +### Protocol Advantages and Limitations + +**Advantages:** +- Reduced contamination risk through elimination of organic solvents and accelerators +- Enhanced mechanical stability during sectioning due to improved particle-matrix adhesion +- Preservation of volatile and thermally sensitive phases through low-temperature processing +- Compatibility with multiple analytical techniques (TEM, STEM, SIMS) +- Improved handling characteristics and reduced sample loss during preparation + + +**Limitations:** +- Particle size restrictions based on sample hardness +- Potential for plucking artifacts in samples with high mineral hardness contrast +- Requirement for specialized temperature control equipment +- Limited long-term storage stability compared to epoxy embedding +- Issues with sulfur reactivity post-sublimation (copper sulfate dendrite) diff --git a/07_troubleshooting.md b/08_troubleshooting.md similarity index 82% rename from 07_troubleshooting.md rename to 08_troubleshooting.md index b11b1f6..d663a8c 100644 --- a/07_troubleshooting.md +++ b/08_troubleshooting.md @@ -5,9 +5,9 @@ title: Troubleshooting Guide ::::{tab-set} -:::{tab-item} Compression +:::{tab-item} Compression -:::{list-table} +```{list-table} :widths: 15 15 30 :header-rows: 1 @@ -15,14 +15,15 @@ title: Troubleshooting Guide - **Causes** - **Solutions** * - inconsistent thickness and shape, wrinkled appearance - - dull knife, excessive feed rate, wrong clearance angle - - reduce cutting speed, increase clearance angle, install sharper knife, reduce feed rate, monitor cutting force + - dull knife, excessive feed rate, wrong clearance angle + - reduce cutting speed, increase clearance angle, install sharper knife, reduce feed rate, monitor cutting force +``` + ::: :::{tab-item} Chatter - -:::{list-table} +```{list-table} :widths: 15 15 30 :header-rows: 1 @@ -30,29 +31,31 @@ title: Troubleshooting Guide - **Causes** - **Solutions** * - regular oscillation marks on sections, "ripping" - - vibration, loose mounting, resonance - - identifying vibration sources, improve isolation, check block mounting stability, lower feed rate/speed + - vibration, loose mounting, resonance + - identifying vibration sources, improve isolation, check block mounting stability, lower feed rate/speed +``` + ::: :::{tab-item} Edge Chipping - -:::{list-table} +```{list-table} :widths: 15 15 30 :header-rows: 1 - + * - **Symptoms** - **Causes** - **Solutions** * - irregular edges, debris in sections - - knife dullness, excessive force, brittle fracture - - lower feed rate, adjust approach angle, verify knife sharpness + - knife dullness, excessive force, brittle fracture + - lower feed rate, adjust approach angle, verify knife sharpness +``` + ::: :::{tab-item} Empty Space/Bubbles - -:::{list-table} +```{list-table} :widths: 15 15 30 :header-rows: 1 @@ -60,17 +63,15 @@ title: Troubleshooting Guide - **Causes** - **Solutions** * - holes in section, sample drop out, chatter - - bubbles inside the molten sulfur - - heat sulfur slowly and evenly, avoid excess movement while embedding + - bubbles inside the molten sulfur + - heat sulfur slowly and evenly, avoid excess movement while embedding +``` ::: - - :::{tab-item} Discoloration - -:::{list-table} +```{list-table} :widths: 15 15 30 :header-rows: 1 @@ -78,8 +79,9 @@ title: Troubleshooting Guide - **Causes** - **Solutions** * - sulfur color change from light yellow to orange - - overheating - - monitor temperature carefully, use temperature controlled heating and consistenct calibration to avoid scorching, keep sulfur temperature below 120°C, do not re-use sulfur, discard discolored sulfur + - overheating + - monitor temperature carefully, use temperature-controlled heating and consistent calibration to avoid scorching, keep sulfur temperature below 120°C, do not re-use sulfur, discard discolored sulfur +``` ::: @@ -124,4 +126,3 @@ title: Troubleshooting Guide ```{figure} ./figures/overheated_annotated_final.png **Representative crystallized sulfur droplets showing correct (left) and overheated (right) samples.** The left droplet displays the expected coloration indicative of properly processed sulfur, while the right droplet exhibits an orange discoloration characteristic of thermal degradation or scorching. Overheating causes partial oxidation or structural changes in the sulfur crystal lattice, resulting in the observed color shift. ``` -::: \ No newline at end of file diff --git a/README.md b/README.md index e52145b..ab20ae8 100644 --- a/README.md +++ b/README.md @@ -40,19 +40,18 @@ Steps to edit locally: - start the jupyter server in one of the terminal windows (`jupyter lab --IdentityProvider.token=512ac78f14e1141db1fac17e8b4099c1e5bc7d589518b38c --ServerApp.allow_origin='http://localhost:3000' --port=8888`) - start MyST in the other terminal window (`myst start`) - edit, commit, and push to `dev` as per usual - - make sure **NOT** to commit your `must.yml` changes! + - make sure **NOT** to commit your `myst.yml` changes! - open a draft pull request into `main` (if one doesn't already exist) and keep pushing your changes to `dev` - this will enable live previews and checks (see below) - - when you're ready, merge pull request into `main`. Note this need to be "final" -- it's best practice to merge thematic content changes together + - when you're ready, merge pull request into `main`. Note this needs to be "final" -- it's best practice to merge thematic content changes together > [!NOTE] > If you don't plan on editing the notebooks, you can skip the `myst.yml` and `jupyter lab` steps above ## Preview draft deployed site -The repo has two github actions to automatically deploy computational sites, for the following two case: +The repo has two GitHub Actions to automatically deploy computational sites, for the following two cases: - Commits to `main` directly - Pull requests into `main` If you followed the instructions above (i.e. working off of `dev` and have an open pull request into `main`), then you should see a `github-actions` bot at the top of your pull request which will keep getting edited. Simply click on the `Inspect` link to see the curvenote staging site, and press `preview` to see the deployed site based on your latest commit. - diff --git a/index.md b/index.md index 5c29cdd..47b075c 100644 --- a/index.md +++ b/index.md @@ -1,5 +1,5 @@ --- -title: 'Sulfur Embedding for Epoxy-Free Ultramictrotomy' +title: 'Sulfur Embedding for Epoxy-Free Ultramicrotomy' short_title: UM methods numbering: heading_2: false @@ -7,29 +7,21 @@ numbering: +++ {"part": "abstract"} -Ultramicrotomy is a well established method for preparation of serial cross-section samples for microscopy and spectroscopy studies. Typical ultramicrotomy protocols begin with embedding the sample in epoxy, which serves as a robust physical support during the sectioning and transfer of sections to slide, stub or grid mounts. For many experiments, the presence of the sectioned epoxy surrounding the actual sample is inconsequential. However, even small amounts of epoxy can present a problem for studies in ultra-high vacuum, or involving high current focused electron beams or involving polymer and hybrid soft-hard materials, due to mechanical instability and chemical alteration of the samples, and contamination of both the sample and the instrument analysis chamber. Sulfur is an alternative embedding medium that can reduce contamination, enhance mechanical stability during sectioning, and yield ultra-thin specimen sections free of any embedding medium. Sulfur embedding has been used successfully for organic macromolecular samples, complex geological samples, but is not yet widely used in materials science. To facilitate broader accessibility and implementation of the technique, we present a detailed protocol for sulfur embedding and ultramicrotomy with example images of ideal conditions and strategies for avoiding artifacts and common procedural challenges. - -Keywords: sulfur embedding, extraterrestrial materials, ultramicrotomy, transmission electron microscopy, sample preparation - +Ultramicrotomy is a well-established method for preparation of serial cross-section samples for microscopy and spectroscopy studies. Typical ultramicrotomy protocols begin with embedding the sample in epoxy, which serves as a robust physical support during the sectioning and transfer of sections to slide, stub or grid mounts. For many experiments, the presence of the sectioned epoxy surrounding the actual sample is inconsequential. However, even small amounts of epoxy can present a problem for studies in ultra-high vacuum, involving high-current focused electron beams, or involving polymer and hybrid soft-hard materials, due to mechanical instability and chemical alteration of the samples, and contamination of both the sample and the instrument analysis chamber. Sulfur is an alternative embedding medium that can reduce contamination, enhance mechanical stability during sectioning, and yield ultra-thin specimen sections free of any embedding medium. Sulfur embedding has been used successfully for organic macromolecular samples and complex geological samples, but is not yet widely used in materials science. To facilitate broader accessibility and implementation of the technique, we present a detailed protocol for sulfur embedding and ultramicrotomy with example images of ideal conditions and strategies for avoiding artifacts and common procedural challenges. +++ +++{"part":"epigraph"} :::{warning} Pre-print This article has not yet been peer-reviewed. -_Updated 2024 August 27_ +_Updated 2026 March 15_ ::: - +++ +++ {"part": "acknowledgements"} - Add your acknowledgments, if any, here. - +++ +++ {"part": "competing interests"} -## Competing Interests - Add your competing interests, if any, here. +++ diff --git a/myst.yml b/myst.yml index c9d8a59..5ac6858 100644 --- a/myst.yml +++ b/myst.yml @@ -3,12 +3,16 @@ version: 1 project: id: msa-em-um-methods-1 title: Ultramicrotomy Methods - description: Sulfur Embedding for Ultramictrotomy. - short_title: UM Methods + description: Sulfur Embedding for Ultramicrotomy. + short_title: Ultramicrotomy Methods keywords: - - Keyword One - - Keyword Two - - Spell Out Acronyms + - ultramicrotomy + - sulfur embedding + - ultrathin sectioning + - TEM sample preparation + - transmission electron microscopy + - astromaterials + - extraterrestrial materials github: https://github.com/msa-em/um-methods jupyter: true # jupyter: @@ -36,13 +40,9 @@ project: name: Anna S.H. Kulenguski corresponding: true email: ahindric@asu.edu - orcid: 0000-0003-2348-8558 + orcid: 0009-0003-5378-0928 affiliations: - - id: ncem - department: School of Earth and Space Exploration - institution: Arizona State University - address: 1151 S Forest Ave, Tempe, Arizona, USA 85287 - ror: 02jbv0t02 + - asu roles: - Conceptualization - Funding acquisition @@ -52,20 +52,22 @@ project: - Writing - original draft - id: rhonda name: Rhonda Stroud + corresponding: true email: rstroud1@asu.edu - orcid: 0000-0001-8338-3323 + orcid: 0000-0001-5242-8015 affiliations: - - id: miller - department: Buseck Center for Meteorite Studies - institution: Arizona State University - address: 1151 S Forest Ave, Tempe, Arizona, USA 85287 - ror: 01an7q238 + - asu roles: - Conceptualization - Software - Validation - Visualization - Writing - original draft + affiliations: + - id: asu + institution: Arizona State University + address: 1151 S Forest Ave, Tempe, Arizona, USA 85287 + ror: 03efmqc40 # funding: # - statement: We thank the Microscopy Society of America for sponsoring this project. # recipients: @@ -78,11 +80,18 @@ project: # recipients: # - cophus abbreviations: - 4D-STEM: four dimensional scanning transmission electron microscopy + 4D-STEM: four-dimensional scanning transmission electron microscopy + EDS: energy-dispersive X-ray spectroscopy + ELNES: electron energy-loss near-edge structure FFT: fast Fourier transform - HAADF: high angle annular dark field - HRTEM: high resolution transmission electron microscopy - M&M: Microscopy and Microanalysis + FIB: focused ion beam + FIB-SEM: focused ion beam scanning electron microscopy + HAADF: high-angle annular dark field + HRTEM: high-resolution transmission electron microscopy + S/TEM: scanning/transmission electron microscopy + SIMS: secondary ion mass spectrometry STEM: scanning transmission electron microscopy + STXM-XANES: scanning transmission X-ray microscopy X-ray absorption near-edge structure + TEM: transmission electron microscopy site: template: book-theme