Specimen Preparation Research Articles

Quality control for single-cell analysis of high-plex tissue profiles using CyLinter.

Tumors are complex assemblies of cellular and acellular structures patterned on spatial scales from microns to centimeters. Study of these assemblies has advanced dramatically with the introduction of high-plex spatial profiling. Image-based profiling methods reveal the intensities and spatial distributions of 20-100 proteins at subcellular resolution in 103-107 cells per specimen. Despite extensive work on methods for extracting single-cell data from these images, all tissue images contain artifacts such as folds, debris, antibody aggregates, optical aberrations and image processing errors that arise from imperfections in specimen preparation, data acquisition, image assembly and feature extraction. Here we show that these artifacts dramatically impact single-cell data analysis, obscuring meaningful biological interpretation. We describe an interactive quality control software tool, CyLinter, that identifies and removes data associated with imaging artifacts. CyLinter greatly improves single-cell analysis, especially for archival specimens sectioned many years before data collection, such as those from clinical trials.

Specimen preparation for atom probe tomography

Abstract In the past 20 years or so, atom probe tomography has gone from a niche technique in physical metallurgy to a well-established method in many parts of materials science and beyond. This is owing to the improvement in both instrumentation and specimen preparation. Given the availability of instruments, successful specimen preparation is often the bottleneck in atom probe projects. In this article, the authors want to give an overview of the types of preparation techniques that are available and which challenges they pose. This includes basic electropolishing, which was dominant until the introduction of focused ion beam-based preparation some 25 years ago. Nowadays, focused ion beam-based preparation methods possibly represent the largest share, as they allow for site-specific specimen preparation, non-conductive specimens and often a higher throughput. They are continuously improved, i. e., through higher current plasma ion columns, cryo preparation for sensitive materials and room temperature liquids and ‘additive’ techniques for nanomaterials. This steadily increases the types of materials that can be analyzed using atom probe tomography.

Flammability and Mechanical Testing of Sandwich Composite for Rolling Stock Structural Applications

Components made of composite materials are being increasingly used in the construction of rolling stock. Currently, the use of components made of composite materials as train structural elements is increasingly being considered. Non-structural components made of composites are most often found inside rail vehicles (e.g., the interior lining), while structural components made of sandwich composite materials can be used for the roof, sidewalls, and underframe constructions. This article provides a description of an innovative sandwich composite developed for a metro’s underframe, as well as the production process and preparation of the composite specimens. The main parts of the work are flammability and mechanical (static and fatigue) tests of the innovative sandwich composite. The scope of the flammability tests included the testing of the fire properties using the radial plate method, the optical density of smoke, and the content of toxic gases. The mechanical strength of the sandwich composite was examined during a flexural (three-point bending) test and a fatigue strength under a given dynamic load. The results presented in the article are very significant, both in terms of flammability and the mechanical strength tests. In order to produce large-size train components, appropriately large patches of component layers of the composite are required; this may pose production problems.

Assessing the Effect of Specimen Preparation Methods on DSR Test Results of Bitumen Using Factorial Design Analysis.

A two-level three-factor factorial design experiment was conducted to study the influences of three critical specimen preparation parameters on the measurement results of bitumen by a dynamic shear rheometer (DSR). The investigated factors were (1) the pre-heating temperature (HT) for manufacturing the specimen, (2) the bonding temperature (BT) onto the rheometer, and (3) the trimming (Trim) operation for preparing the specimen after bonding. The analysed data were the measured shear modulus |G*|, phase angle δ, and the characteristic temperatures of bitumen's specific stiffness TX with corresponding phase angle δTX according to the European standard EN 14770:2023. Five types of bitumen were tested, including three penetration grades and two modified bitumen specimens (with polymer and wax additives). In addition, a repeatability evaluation of the test results was conducted. We found that the trimming operation for preparing the specimen has a noticeable impact when using smaller plates (PP08) for the DSR measurement. At higher test temperatures when using larger plates (PP25), the trimming operation does not significantly impact the measured parameters, in contrast to the HT and BT. Except for bitumen type 70/100, modified binders are more susceptible to variation in the analysed parameters than unmodified ones. The three-way interaction Trim:BT:HT tends to cause relatively little variation in measured data. Interactions between two factors Trim:BT, Trim:HT, and BT:HT contribute more to the fluctuation in δ value than in TX and |G*|. The variation employed in this study affects the test repeatability of wax-modified bitumen significantly; however, for unmodified binders the repeatability of TX and δTX are within 0.4-2.1 °C and 0.3-3.1°, respectively.

Dynamics of Amphibian Pathogen Detection Using Extended Museum Specimens.

Natural history collections have long served as the foundation for understanding our planet's biodiversity, yet they remain a largely untapped resource for wildlife disease studies. Extended specimens include multiple data types and specimen preparations that capture the phenotype and genotype of an organism and its symbionts-but preserved tissues may not always be optimized for downstream detection of various pathogens. Frogs are infected by an array of pathogens including Batrachochytrium dendrobatidis (Bd), Ranavirus (Rv), and Amphibian Perkinsea (Pr), which provides the opportunity to study differences in detection dynamics across tissue types. We used quantitative PCR protocols to screen two tissue types commonly deposited in museum collections, toe clips and liver, from two closely related host species, Rana catesbeiana and Rana clamitans. We compared Bd, Rv, and Pr infection prevalence and intensity between species and tissue types and found no significant difference in prevalence between species, but Bd intensity was higher in R. clamitans than R. catesbeiana. Toe tissue exhibited significantly higher Bd infection loads and was more useful than liver for detecting Bd infections. In contrast, Rv was detected from more liver than toe tissues, but the difference was not statistically significant. Our results support the use of extended specimen collections in amphibian disease studies and demonstrate that broader tissue sampling at the time of specimen preparation can maximize their utility for downstream multipathogen detection.

Clinical Test Comparison for Improved Diagnosis of Mucormycosis

Abstract Mucormycosis is a rapidly progressive and invasive fungal infection caused by members of the order Mucorales. Proper treatment relies on a timely diagnosis. However, traditional fungal culture takes several days to weeks and has low sensitivity for recovering Mucorales, as these organisms are sensitive to specimen preparation, including tissue homogenization, and low storage temperatures. More rapid and sensitive diagnostic methods are needed to promptly detect infection. Our laboratory previously developed a real-time PCR assay that detects the most common genera associated with mucormycosis to help improve diagnosis. The assay was analytically validated for a variety of specimen types including serum, tissue (both fresh and FFPE), respiratory secretions and body fluids. Little is known about the agreement between our laboratory developed PCR and other detection methods such as culture or sequencing. We reviewed 6 years of laboratory data to assess overall PCR positivity rate, positivity rate across sample types, and agreement with culture and/or pan-fungal sequencing. We also calculated turnaround time to results to gauge the diagnostic value of Mucorales PCR. Test comparisons with fungal culture and/or pan-fungal sequencing were performed if testing was ordered within 30 days of each other. In our study, we reviewed 3,804 total PCR orders for 2,195 patients. The overall positivity rate of Mucorales PCR orders was 3.81% and varied among different sample types, with fresh tissue and body fluid having the highest positivity rates (18.11% total tissue (33.3% lung tissue) and 10.94% all body fluid (12.5% pleural fluid)) and sputum and CSF having the lowest positivity rates (<1% positivity for both). In a cohort of 428 patients with fungal culture and/or sequencing also performed, 29 patients tested positive for Mucorales by at least one method. PCR detected Mucorales on average 7.83 days sooner than fungal culture (average time to positive result: 3.65 vs. 11.47 days). In patients with culture- or sequencing-confirmed Mucorales infection, PCR had a 100% agreement when comparing shared samples from the same source and collection time. Mucorales PCR was also able to detect infection in 10 (34.5%) other patients who were culture-negative for Mucorales. Our findings suggest that PCR could be a valuable diagnostic tool for suspected mucormycosis, especially tissue invasive disease. PCR increased case detection by 34.5% with a faster turnaround time than culture with lesser cost than sequencing. The main limitation of our study was lack of access to patient information to assess pretest probability of Mucormycosis.

Lift-Out Specimen Preparation and Multiscale Correlative Investigation of Li-Ion Battery Electrodes Using Focused Ion Beam-Secondary Ion Mass Spectrometry Platforms.

Advanced characterization is paramount to understanding battery cycling and degradation in greater detail. Herein, we present a novel methodology of battery electrode analysis, employing focused ion beam (FIB) secondary-ion mass spectrometry platforms coupled with a specific lift-out specimen preparation, allowing us to optimize analysis and prevent air contamination. Correlative microscopy, combining electron microscopy and chemical imaging of a liquid electrolyte Li-ion battery electrode, is performed over the entire electrode thickness down to subparticle domains. We observed a distinctive remnant lithiation among interparticles of the anode at the discharge state. Furthermore, chemical mapping reveals the nanometric architecture of advanced composite active materials with a lateral resolution of 16 nm and the presence of a solid electrolyte interface on the particle boundaries. We highlight the methodological advantages of studying interfaces and the ability to conduct high-performance chemical and morphological correlative analyses of battery materials and comment on their potential use in other fields.

Streamlined chemical fixation method for morphological investigation of Candida albicans with scanning electron microscopy

This method describes the preparation of Candida albicans specimen for scanning electron microscopy (SEM), starting from specimen collection to obtaining SEM images. Our approach involves preserving cellular structure and preventing cell degradation through chemical fixation and post-fixation without Osmium tetroxide (OsO4), followed by air dried specimen placed onto Si substrate. Additionally, the method outlines the steps for mounting the prepared specimen onto SEM sample holder aluminum stubs, followed by coating with an ultrathin gold layer via thermal evaporation to enhance specimen/substrate/stub conductivity. The specimens are then mounted in the SEM sample holder. This step-by-step procedure demonstrates successful morphological analysis of C. albicans, providing a comprehensive and effective specimen preparation for SEM analysis.•Effective preparation of Candida albicans specimens for SEM analysis, preserving cellular structure.•Step-by-step specimen mounting and gold coating for enhanced detailed SEM imaging.•Chemical fixation of yeast cells for electron microscopy visualization.

A-324 Performance and Turnaround Time of Plasma Lactate Assay on POCT, Blood Gas Instrument, and Chemistry Analyzer

Abstract Background Time is a critical factor of plasma lactate testing in the management of patients with sepsis. To best serve these patients, we must optimize testing methods to provide a turnaround time (TAT) that guides efficient patient care. Point of care tests (POCTs) provide rapid results for emergency department and medical intensive care unit but are not feasible for all hospital settings. Laboratory testing methods on an automation line provide greater efficiency but result slowly due to need of specimen preparation and longer analytical time. The goal of this study is to provide detailed TATs of plasma lactate testing among the methods of POCT, chemistry analyzer in an automation line, and blood gas analyzer. Methods Plasma lactate testing was performed by three methods, GEM Premier ChemSTAT (POCT in ER only), Beckman AU5800 (chemistry analyzer), and Radiometer ABL800 (blood gas instrument). The TAT of lactate for each method was measured and include the time from specimen collection to laboratory receipt (except for POCT method), and time from specimen receipt to resulting (including specimen preparation for Beckman AU5800). Results The average TATs of lactate testing for each method in all hospital sites are detailed with respect to each event and subcategorized (Table 1). The average TAT of 12.0 min on Radiometer ABL800 is higher than the TAT of 2.5 on GEM Premier ChemSTAT method but is greatly reduced compared to the TAT of 35.4 min on Beckman AU5800. Conclusions Radiometer ABL800 method could be the optimal laboratory method for plasma lactate testing fulfilling the TAT (average 12 min), with less test cancellation due to specimen stability issue, and combines testing with blood gases. Using three Radiometer ABL800 instruments, we manage the performance of around 70 plasma lactate tests daily.

Development and testing of a novel vibratory compaction instrument considering its natural frequency.

This study aims to develop a novel vibratory compaction instrument designed for the preparation of large triaxial soil specimens, offering an efficient alternative to the traditional manual specimen preparation process. However, when the motor frequency closely aligns with the natural frequency of the structure, severe resonance phenomena may occur. This paper employed finite element modal analysis, experimental modal analysis, and operational modal analysis techniques to conduct a multi-objective optimization of the structure, aiming to improve the dynamic characteristics. First, the natural frequency and mode shapes of the vibratory compaction instrument were determined via finite element technology. Subsequently, the optimization of this instrument was conducted by integrating the Kriging surrogate model with the Non-dominated Sorting Genetic Algorithm II (NSGA-II). Finally, the steel column underwent a modal test using impulse stimulation to ascertain its natural frequency, and the stochastic subspace identification (SSI) approach was employed to conduct an operational modal analysis of the vibratory compaction instrument to determine its modal characteristics. The experimental findings corroborated the conclusions obtained from the finite element analysis, validating the precision of the finite element modal analysis and the rationality of the optimized structure. The research results are of significant importance for improving the preparation efficiency of large triaxial specimen, contributing to advancements in soil mechanics testing.

A simplified methodology for assessing the interface of CFRPs with fibre push-out testing

This work proposes a simplified methodology for examining the interface of fibre reinforced composites with fibre push-out testing. Demonstrated in a carbon fibre (CF) reinforced polymer (CFRP) case study, it examines the interface of a commercial epoxy with the 31E, 50C, and F0E T700 CFs. Atomic force microscopy, attenuated total reflection – Fourier transform infrared spectroscopy, and scanning electron microscopy are used to characterise the fibres, and the CFRP samples are manufactured with wet winding. To create trenches of suitable thickness within the bulk composite, femtosecond-laser processing is proposed as a localised material subtractive method, facilitating the fibre push-out testing with the flat nanoindenter tip. The results highlight the influence of the CF sizing agent on the CFRP interface, and overall, a methodology is defined that simplifies the specimen preparation for assessing the interface of FRPs. This advancement holds promise for a broader adoption of such tests from both academia and industry.

Minimizing ice contamination during specimen preparation for cryo-soft X-ray tomography and cryo-electron tomography

Cryo-soft X-ray tomography (cryo-SXT) is a newly developed technique for imaging 3D whole cells in nearly native states. Cryo-SXT users require the preparation of numerous cryo-sample grids to use the allocated beamtime to study cellular phenomena under various conditions. Therefore, it is important to promptly prepare cryo-sample grids as efficiently and carefully as possible to minimize ice contamination on the frozen sample grid. In this study, we designed a cryo-multi-grid-box storage system, which includes a shell, funnel holder, and multi-grid-box container. Our system not only increases the number of cryo-sample grids that can be temporarily stored but also reduces the frequency of cryo grid-box container transfers, thus decreasing the probability of forming ice on the grid. We have also applied this system to A549 cryo cell grid preparation. The correlative images from cryo light microscopy and cryo-SXT showed that limited ice had formed on the grid when preparation was performed using our system. Additionally, 3D images of mitochondria with the lamellar shape of the cristae could be observed in our cryo-SXT results. Our cryo-multi-grid-box storage system can be used for cryo-SXT and cryo-electron tomography (cryo-ET) applications.

A refined plan-view specimen preparation technique for high-quality electron microscopy studies of epitaxially grown atomically thin 2D layers

The structural studies of two-dimensional (2D) van der Waals heterostructures and understanding of their relationship with the orientation of crystalline substrates using transmission electron microscopy (TEM) presents a challenge in developing an easy-to-use plan-view specimen preparation technique. In this report, we introduce a simple approach for high-quality plan-view specimen preparation utilizing a dual beam system comprising focused ion beam and scanning electron microscopy.To protect the atomically thin 2D heterostructure during the preparation process, we employ an epoxy layer. This layer serves as a protective barrier and enables the creation of a TEM specimen comprising a thin substrate fragment with an overgrown 2D structure covered by a thin, electron-transparent epoxy layer. The coexistence of both 2D layers and substrate is essential for investigating the relative crystallographic orientations between the grown 2D structures and the substrates. The thickness of the specimen is monitored using low-voltage scanning electron microscopy.We apply this technique to prepare plan-view specimens of 2D germanium-antimony-telluride (GST) on Si and hexagonal boron nitride (h-BN)/epitaxial graphene (EG) heterostructures grown on 6H-SiC substrates. The grain-like atomic structure observed in the 2.2 nm thick GST layer on Si substrate provides evidence of the mosaicity of GST during the early stages of epitaxial growth. H-BN/EG on 6H-SiC structural studies indicate a rotation of h-BN/EG around the 6H-SiC[0001] axis by an angle of 30°. The observed BN particles with sizes in the nanometer range on top of the sample have the wurtzite lattice type and random orientation.The developed specimen preparation technique offers a powerful tool for TEM studies of atomically thin layers on crystals. Its simplicity and ability to provide valuable insights into the in-plane relationships between 2D structures and crystalline substrates make it a promising complement to grazing incident X-ray diffraction.

Hybrid Effect of Metakaolin and Nano-Silica on Mechanical and Durability Parameters of Mortar

The incorporation of pozzolanic materials such as nano silica and metakaolin in cementitious materials has gained significant attention in recent years due to their potential to improve the mechanical and durability properties of cement-based composites. This thesis investigates the hybrid effect of nano silica and metakaolin on the mechanical and durability properties of cement mortar. The research work involved the preparation of cement mortar specimens with varying proportions of nano silica and metakaolin, which were then subjected to a series of mechanical and durability tests. The mechanical tests included compressive strength, while the durability tests included water absorption and elevated temperature test. The study showed that the addition of both nano silica and metakaolin to cement mortar resulted in a significant improvement in the mechanical properties of the mortar. Additionally, the hybrid effect of nano silica and metakaolin resulted in enhanced durability of the cement mortar, with a significant reduction in water absorption and improved temperature resistance. The findings of this study will have significant implications for the development of sustainable and durable cementitious materials. The use of pozzolanic materials such as nano silica and metakaolin can reduce the consumption of cement and the associated carbon footprint while also improving the durability and long-term performance of cement-based composites. Overall, this study suggests that the incorporation of nano silica and metakaolin can lead to significant improvements in the mechanical and durability properties of cement mortar, making it a promising material for use in various construction applications.

Aspergillus aureoterreus, A. pseudonomiae, A. siamensis, and A. flocculosus-ochraceopetaliformis as Four New Pathogens of Eye Infection in DNA Barcoding Markers

Background: The use of a morphological method to determine Aspergillus species in routine laboratory investigation has always been challenged with low sensitivity and low specificity. Time-consuming specimen preparation and insufficient expertise are the crucial reasons. DNA barcoding markers have been introduced for identification as another gold standard method. Objective: To investigate DNA barcoding markers in identifying Aspergillus strains isolated from patients with eye infection. Methods: This study extracted 12 DNA samples of 12 Aspergillus strains. DNA barcoding genes, i.e., ITS, betatubulin (BT2), calmodulin (CAL), and actin (ACT) genes, were amplified and sequenced by polymerase chain reaction and Sanger's sequencing methods. All gene sequences were compared to those of the type or reference species collected from public databases. The species were identified with phylogenetic analyses in free applications from public websites. Results: ITS, BT2 and CAL, and ACT identified 5, 6, and 3 species and 3, 2, and 1 species complex of Aspergillus, respectively. DNA barcoding with multi-locus sequence typing revealed four new pathogens of human eye infection, including A. aureoterreus, A. pseudonomiae, A. siamensis, and A. flocculosus-ochraceopetaliformis. Conclusion: The DNA barcoding markers identified Aspergillus species with better specificity than the conventional method. The markers and techniques need to be used as tools for routine Aspergillus identification.

Influence of fines and gravel particles on strength-dilatancy of river sand: Effect of depositional conditions

Understanding the effects of low plastic fines and gravel contents on the mechanical behavior of sandy soils in terms of the strength-dilatancy relationship is crucial for various geotechnical applications. To gain deeper insights, an extensive series of direct shear tests were performed on two distinct types of mixtures: sand-fines mixtures and sand-gravel mixtures. The study systematically varied Fc/Gc content from 0% to 30%, using dry and wet methods, at a medium density (Dr = 50%) and a range of normal stresses (σn = 100–300 kPa). The experimental findings provide remarkable trends. For dry pluviated specimens, an increase in fines content led to a decrease in the shear strength of the mixtures. Conversely, the wet deposited specimens exhibited an opposite tendency, where higher fines content induced larger shear strength. Notably, the incorporation of gravel substantially improved the mechanical performance of the sand mixtures, regardless of the preparation method employed. Dry pluviated specimens generally exhibited higher shear strength than that of the wet deposited ones for the same Fc/Gc. A developed set of equations has been suggested to predict the strength-dilatancy relationships for all-tested materials. These equations take into account the impacts of fines and gravel contents and both specimen preparation methods.

Influence of dimensional deviation and thickness non-uniformity on the small-size specimen tensile results of CLF-1 steel

Tensile testing of small-size specimens has been widely used to evaluate the influence of neutron irradiation on the mechanical properties for structural materials of fusion reactors. In recent years, researchers have been trying to standardize small-size specimen tensile test method. In the present paper, we have investigated the influence of dimensional deviation and thickness non-uniformity on the tensile results of small-size specimen by both experiments and finite element simulations, based on one of the candidate structural materials of fusion reactor, i.e. CLF-1 steel. It was found that under the conditions of small grain size, small metallurgical defect size and suitable specimen preparation, the tensile results of SS-J3 specimen could be close to the results of large-size specimen. Slightly higher yield and ultimate strengths for small specimens may be caused by the surface treatment of the wheel grinding. The repeatability of ultimate strength was better than that of yield strength, and the repeatability of elongation was worse than that of strength. For tensile tests on eight SS-J3 specimens, the maximum difference in total elongation was about 3 %. With ±0.1 mm deviation of thickness and parallel width, the maximum variation in total elongation was about 1.8 %. The ductility properties were sensitive to specimen's thickness non-uniformity, the measured uniform and total elongations would obviously decrease even with 0.01 mm thickness non-uniformity.

SIFAT MEKANIS KOMPOSIT MATRIKS ALUMINIUM BERPENGUAT ABU JERAMI DENGAN METODE METALURGI SERBUK

The powder metallurgy method is one method for making metal matrix composite products. This method provides researchers with material engineering.This study aims to determine how the effect of variations in mixing time and sintering holding time affects the hardness and compressive strength of composites made of aluminum powder and straw ash. The composition of this composite is 90%wt Aluminum and 10%wt straw ash. Preparation of specimens using powder metallurgy method. Mixing time variations were applied for 120, 150 and 180 minutes with the dry mixing method. Variation of sintering holding time was applied for 60, 120 and 180 minutes at 550°C.The mechanical test carried out are hardness test and compression test. The results showed that the optimum hardness value was found in the specimens with a mixing time of 150 minutes and sintering holding time of 180 minutes with an average hardness of 96 HRF. The optimum compressive strength is found in specimens with a mixing time of 150 minutes and sintering holding time of 120 minutes with an average compressive strength of 101.28 MPa. It can be concluded that mixing time and sintering holding time affect the hardness and compressive strength of the metal matrix composite.

An efficient and robust method for evaluating the low-temperature performance of asphalt binder based on DSR testing

The BBR test has received growing concerns regarding its applicability to field studies and special materials due to the large amount of long-term aged asphalt binder used in preparing asphalt binder beams and the time-consuming beam preparation and testing process. In recent years, the application of a dynamic shear rheometer (DSR) to investigate the low-temperature performance of asphalt binder has attracted widespread attention because it uses significantly less asphalt binder for testing and allows for easy specimen preparation. The objective of this study was to develop an efficient and robust method to evaluate the low-temperature performance of asphalt binder based on DSR testing. Frequency sweep tests were performed on twelve asphalt binders at temperatures ranging from 0 to 25 °C using the 8-mm parallel plate geometry. Two conversions were applied to frequency sweep test data in order to predict the flexural creep stiffness and m-value of asphalt binder, namely the conversion from frequency domain to time domain and the conversion from shear loading to bending loading. The 1S2P1D model was employed to construct the master curves of storage modulus and loss modulus. Afterwards, the flexural creep stiffness was predicted using the shear creep compliance master curve model, which was derived from the 1S2P1D model using the inverse Laplace transform, and the Poisson’s ratio, which was assumed to be constant. It was found that the predicted values of S(60) and m(60) were comparable to the counterparts measured by the BBR, which well demonstrated the feasibility of the proposed DSR-BBR conversion method. In comparison to existing analysis methods, the proposed conversion method strictly complied with the linear viscoelastic theory, and it enabled efficient and robust predictions because the prediction model of flexural creep stiffness had the same parameters as those of the 1S2P1D model, thus avoiding multiple data fittings.

On the preparation and mechanical testing of nano to micron-scale specimens

Over the past two decades, means allowing to probe experimentally the mechanical behavior of materials specimens only a few micrometers or less in diameter have multiplied, largely owing to the widespread adoption and versatility of focused ion beam (FIB) milling for the preparation of small-scale test specimens. Despite its remarkable capabilities, the ion bombardment that is employed during FIB milling operations does not leave machined surfaces unscathed: it may induce a series of alterations in the near-surface microstructure of materials, which vary in severity depending on the material being milled, or milling parameters, or the type of ion used. These alterations in turn can strongly influence the local behavior, and as a result measured mechanical properties, of the milled material. In the first part of this manuscript, we review the different forms that FIB-induced microstructural alterations can take and their influence on small-scale mechanical test results. In the second part, we present alternative strategies that have been used to circumvent the issue. These include the use of entirely different small-scale sample fabrication processes, as well as approaches that do use FIB-milling but put effort in the test design to minimize or avoid the formation of FIB-induced defects in regions where micromechanical test data are collected. The advent of such methods can enhance our understanding of FIB-induced defects on the mechanical behavior of microsamples by comparing the results with those from ion-milled samples. This, in turn, should improve our ability to interpret test data when FIB-milling is the only method available for microsample production.