Holotomographic microscopy (HTM) measures the refractive index (RI) tomograms of living cells and tissues in three dimensions. The ability to observe biological processes at high spatial and temporal resolution opens uncharted territories for cell biologists, however, current HTM devices have a limited throughput. We show here the first automated multi-well plate-compatible HTM device, the CX-A. Thanks to state-of-the-art environment control and a new type of autofocus, the CX-A can record multiple conditions in parallel over large fields of view, while its software EVE supports automated single-cell segmentation and quantification. This opens the door to new applications for HTM, from drug screening to systems biology.

Previous studies of nickel hydroxide (Ni(OH)2) powders have shown that either heating or mechanical grinding can result in complete de-hydroxylation, leading to conversion to nickel oxide (NiO). In both cases, this process appears to occur in one stage, without evidence for any intermediate compounds being formed. During studies of Ni(OH)2 powders for applications in the positive electrodes of Ni metal hydride (NiMH) rechargeable batteries, using transmission electron microscopy (TEM), we have observed significant changes caused by exposure to the highly energetic electron beam used for imaging and analysis. It is shown here, using electron energy-loss spectroscopy (EELS), that de-hydroxylation under electron irradiation occurs in two stages, with nickel oxy-hydroxide (NiOOH) being formed at the intermediate stage.

X-ray diffraction (XRD) is a fingerprint technique for the analysis of atomic and molecular structures of crystalline materials, from polymers and plastics, through to structural composites and biomaterials. These all have crystallographic phases in the nanostructure, which greatly influence the macro properties of the material—from insulin and hemoglobin to semiconductors and solar cells. Here, we look at how XRD analysis using a small- and wide-angle X-ray scattering (SAXS/WAXS) system under full vacuum brings the possibility of crystallographic sample characterization, with temperature and environmental control, direct to the laboratory, and how this improves the workflow for phase identification.

The brain, a lung, a tumor: each contains a complex array of cell types arranged in an intricate spatial pattern that is critical to their role within the tissue. While work is underway to create an atlas of cellular diversity across tissues, traditional methods either have sufficiently high multiplexing capacity but lack spatial context, or they provide spatial information but lack the multiplexing capacity to identify subtle variations in cell type or state. This limitation precludes a comprehensive view across a whole tissue, including rare cells, as they exist within a tissue of interest. Since understanding how the molecular and cellular organization of a biological tissue contributes to its functionality is fundamental to developing treatments that target the cell biology underlying diseased states, improved tools that map biological systems with both high spatial resolution and high multiplexing capacity promise to deliver new, clinically relevant insights into the functional composition of biological systems. The recently developed multiplexed error-robust fluorescence in situ hybridization (MERFISH) method provides information about gene expression at subcellular resolution and reveals cell type, state, and cell-cell interaction by quantifying the expression and exact location of hundreds to thousands of transcripts within intact tissues. MERFISH represents a major advancement toward directly interrogating rich cellular heterogeneity and is being applied to gain insight into the biological complexity within and around us.

This paper presents a proof of concept for the discrimination of several nanoparticle populations mixed in consumer products. The methodology proposes correlation of AFM, SEM, and EDS data to obtain structural and chemical information on each particle in a mixed population. To this end, emphasis is placed on sample preparation, imaging specifications for each instrument, and data correlation with adapted software.

Understanding of a disease and its treatment relies heavily on visualizing cells, tissues, and organs, making micro- and macro-imaging modalities integral to the biomedical community. Macro imaging modalities such as magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound are still the mainstay of whole-body imaging because of their non-invasive nature and live image feed. However, they fail to deliver cellular details required to understand a disease or effectiveness of a treatment. On the opposite end of the spectrum, benchtop microscopes and histology can deliver excellent cellular resolution, allowing one to visualize cellular and intracellular interactions to better understand a disease, its progression and/or the outcome of therapies. However, to look at the cells, the tissue needs to be resected, fixed, processed, and stained before imaging. This whole process of visual confirmation may take hours to days. Deciding on these two imaging modalities always involves a compromise, either in time, resolution, or invasiveness. Confocal laser endomicroscopy (CLE) fills a unique niche between these macro- and micro-imaging modalities as it facilitates non-invasive to minimally invasive in vivo image acquisition in real time. In this two-part series we first examine the equipment available for CLE, and in the second part we will explore several applications of the technology.

In-situ transmission electron microscopy (TEM) provides an avenue to explore time-dependent nanoscale material changes induced by a wide range of environmental conditions that govern material performance and degradation. The In-situ Ion Irradiation TEM (I3TEM) at Sandia National Laboratories is a JEOL 2100 microscope that has been highly modified with an array of hardware and software that makes it particularly well suited to explore fundamental mechanisms that arise from coupled extreme conditions. Examples pertaining to multibeam ion irradiation, rapid thermal cycling, and nanomechanical testing on the I3TEM are highlighted, along with prospective advancements in the field of in-situ microscopy.

In correlative light and electron microscopy (CLEM) workflows, identifying the same sub-cellular features in tissue by both light (LM) and electron microscopy (EM) remains a challenge. Furthermore, use of cryo-fixation for EM is desirable to capture rapid biological phenomena. Here, we describe a workflow that incorporates cryo-confocal laser scanning microscopy into the CLEM process, mapping cells in brain slices to re-image them with serial section scanning electron microscopy (ssSEM) array tomography. The addition of Airyscan detection increased the signal-to-noise ratio (SNR), allowing individual spines in thick frozen tissue to be visualized at a sufficient spatial resolution, providing a new tool for a CLEM approach to capture biological dynamics.

Sideband Kelvin probe force microscopy (KPFM) uses the intermodulation of an electrostatic drive force and a mechanical drive force to upconvert the electrostatic frequency to the first flexural resonance, where the high-quality factor of the resonance yields a more sensitive measurement. The sideband KPFM signal is calculated using a local interaction between the tip apex and the sample, rather than a total interaction between the cantilever and the sample, improving the spatial resolution over other technique variations. This paper covers the sideband KPFM details, including trade-offs and imaging of semi-fluorinated alkanes, such as F14H20, and self-assembled molecular nanostructures.

Providing high-quality electron images and hyperspectral X-ray maps is a focus of many modern electron microscopy laboratories. Nevertheless, further image processing and annotations are often needed to prepare them for publications and reports. For multi-user facilities, accessibility to processing software can be a limitation either through license costs or availability of processing stations. Open-source software running on multiple platforms allows for post-acquisition data processing in-lab or on user-owned devices. We developed Probelab ReImager to supersede our vendor-supplied acquisition software's exportation by being efficient and highly customizable. This article describes its main features and capabilities.

Live-cell fluorescence microscopy is an indispensable tool in biomedical and biophysical research, allowing the study of dynamic processes in living systems. Recent developments have given rise to high-end imaging systems capable of live-cell imaging on the nanometer scale. These instrumentation developments have, in turn, increased the demand for enhanced fluorophores to label target proteins within living cells. abberior offers solutions on both fronts. abberior LIVE dyes have higher cell permeability and improved specificity compared to conventional labels, making long-term live-cell imaging at extremely low probe concentrations possible. These low nanomolar working concentrations minimize cell toxic effects. abberior stimulation emission depletion (STED) microscopes offer super-resolution down to ~30 nm with minimal light exposure to the sample. This unique combination takes live-cell imaging to the next level and will make super-resolution imaging of dynamic subcellular processes a routine, easy-to-use visualization method, paving the way for fundamental biological discoveries. At the extreme, minimal emission fluxes (MINFLUX) microscopy offers molecular resolutions of a few nanometers in unprecedented temporal regimes.

Electron microscopy has enabled atomic resolution imaging of matter. However, unlike optical spectroscopic imaging, traditional electron microscopes provide limited spectroscopic information in terms of their energy resolution. Only recently, owing to advances in monochromated STEM-EELS, have transmission electron microscopes (TEMs) been able to attain a high energy resolution. We recently proposed combining spectrally selective photoexcitation with HRTEM to achieve sub-nanometer scale optical imaging, a technique we called photoabsorption microscopy using electron analysis (PAMELA). To realize PAMELA-TEM experimentally, we constructed a TEM holder with an optical feedthrough, capable of photoexciting materials with different wavelengths. In this article, we describe our process for designing and fabricating an optical TEM specimen holder, highlighting important aspects of the design.

The development and implementation of high-stability monochromators in state-of-the-art aberration-corrected scanning transmission electron microscopes has enabled materials characterization with an energy resolution as good as 3 meV. This allows the vibrational modes, which would otherwise be obscured by the energy spread of the electron beam, to be probed with very high precision in molecular materials. Since the vibrational energies depend on the weight of the atomic nuclei, vibrational spectroscopy can distinguish isotopes whose only difference lies in their neutron content. This opens up isotopic analysis and mapping in transmission electron microscopy as two important new research areas. Here, we review the monochromated electron energy loss spectroscopy (EELS) instrumentation, discuss optimal methods for probing beam-sensitive materials without destroying them, and review key nanoscale isotope-resolved results.

We describe an experimental approach for achieving an optimal black background for scanning electron photomicrographs of small samples with elaborate and intricate structures. Specimens of the highly ornate, 66-million-year-old chorate dinoflagellate cyst species Cannosphaeropsis franciscana were selected as the subject of this study. Photomicrographs collected following standard aluminum stub surface placement were compared to those taken of specimens mounted using a novel pin-and-pedestal method. This simplistic mounting technique minimizes the need for post-production image editing and extraneous background removal.

With the growing applications of temporally resolved electron microscopy for probing basic phenomena and reducing beam-induced damage, a multifaceted introduction to the field of ultrafast transmission electron microscopy is provided. This primer includes techniques and equipment as well as implementation perspectives. Historical developments and recent technical advances will provide insight into ultrafast capabilities for research as well as educate electron microscopists on the general techniques. This technology review also includes applications enabled by ultrafast techniques using various sample stimuli from multidisciplinary fields.

The polarizing light microscope (PLM) is one of the most versatile tools in the laboratory, permitting the analyst to characterize and identify a very large variety of different materials in addition to determining crystalline polymorphs. Most research-grade PLMs allow various adjustments to align the microscope for centration of components to the optical axis. The Olympus BHSP PLM allows three of the four objective mounts in the rotating objective nosepiece to be centered by using two custom centering wrenches that are supplied with the microscope when purchased new. The same wrenches are additionally used to adjust centering of the stage. Over time, the wrenches can become lost or misplaced, and the wrenches are rarely provided with the sale of a used BHSP microscope. Replacement centering wrenches are often needed. Appropriately sized jeweler's screwdrivers can be used, but these do not remain in place and attached to the nosepiece or stage centering screws while focusing or manipulating other parts of the microscope during the centration procedure. This article provides steps in making suitable replacement centering wrenches.

Transmission electron microscopy (TEM) offers significant potential for studying chemical changes that occur in a battery electrode at a spatial resolution approaching atomic dimensions, provided a representative specimen can be prepared. The purpose of this paper is to show that it is possible to examine an electrode in the charged condition using TEM, while avoiding major changes in chemistry from specimen preparation. We also demonstrate the power of electron energy-loss spectroscopy (EELS) for phase identification at high spatial resolution. The origin of “fading” of an electrode after repeated charge-discharge cycling is also discussed.