Fluorescence in vivo endomicroscopy (FIVE) is a state-of-the-art endoscopy technique used to image tissue interactions and molecular events in a cell. In Part 1 of this series, the history, types of confocal laser endomicroscopy (CLE), and limitations of the technology were discussed. In Part 2 of the series, we describe several applications of FIVE technology, including advances in cancer, gastrointestinal, liver, rectal mucosal barrier function, acute lung injury, and brain imaging. Future perspectives for the technology are also presented.

Each year, the Microanalysis Society (MAS) bestows several major awards during the Microscopy & Microanalysis annual meeting. Regrettably, there is never enough time on stage to detail these awardees’ contributions to the science of microanalysis and the Society, which we hope to rectify here and in future articles. The shift of the 2020 M&M meeting to a virtual format meant that our awardees did not receive their accolades on a physical main stage. These recipients were selected for recognition by a variety of mechanisms, of which many Society members may not be aware. For example, the MAS Awards Committee solicits nominations for annual Outstanding Paper Awards from M&M meeting attendees and symposium organizers via a simple webform (https://the-mas.org/mm-outstanding-paper-recommendation/), whereas the Duncumb Award requires a full nomination package from Society members, and other awards are selected solely at the discretion of the MAS President or Council. Additional description of the various MAS awards, including lists of previous award winners, can be found at https://the-mas.org/awards.

The structure and organization of cells within organs is essential to their function, but nowhere in the body is this more spectacular than the brain. There, sprawling, snowflake-like neurons have grown into a precise arrangement, reaching out to neighboring cells to form neural circuits. Communication within neural circuits, made possible by spatial positioning, forms the basis of our physiology. Recently, a high-resolution cell atlas generated by MERFISH (multiplex error-robust fluorescence in situ hybridization) technology has mapped this spectacular organ with unmatched resolution, depth, and scale. The atlas catalogs cells as they exist in the intact biological system and will allow us to learn more about rare cell types and sparsely expressed cell signaling receptors fundamental to health and disease.

This article reviews the use of portable microscopes in published science from Antonie van Leeuwenhoek's letters to the Royal Society to a recent paper on the phylogeny of the phorid fly by Hartop et al. [1]. The portability of a pocket microscope is a clear benefit, yet bulky compound microscopes remain the mainstay of field research and diagnosis. There is a growing interest in the impact of climate change, which drives researchers to investigate the smallest, most fragile life in remote places such as Antarctica, Mount Everest, and the Amazon Rainforest. Are pocket microscopes ready to produce publication-standard images?

For materials characterization, obtaining insight through 3D imaging has been extremely helpful in improving our understanding of complex systems. X-ray computed tomography (CT) has proven to be an extremely useful technique in this field, allowing for non-destructive interrogation of samples of all types across many length scales. In this article we look at how micro-CT is moving from static 3D imaging into the realm of 4D data collection through dynamic CT, where CT data are collected on samples undergoing change in an uninterrupted matter.

Snowflakes are a natural subject for microscopy, but the conditions under which they form, and the minutes-long working time imposed by sublimation, are substantial technical barriers to field microscopy of snow crystals. Building on the work of earlier pioneers, we developed a portable, actively cooled, semi-automated microscope system capable of making z-stacked images of individual snowflakes and small groups of crystal specimens at ultrahigh resolution. Field trials of the system in Alaska and Canada produced encouraging results and also suggested directions for further improvement.

Modern electron microscopy permits scientists to study the fine detail of cells and tissues, using both two-dimensional and three-dimensional imaging modalities. However, achieving optimal preservation of ultrastructure for a variety of biological samples remains a challenge. Here, we describe practical methods to preserve the fine structure of mouse skeletal muscle and sciatic nerve and to obtain high-resolution images of mitochondria in cultured cells, flow-sorted T-cells, and mouse urothelium. We also propose an effective and economical workflow for three-dimensional electron microscopy in the context of a microscopy core facility.

Peripheral nerves in vertebrate systems have been studied for centuries. Nerve anatomy, including the connective tissues that cushion nerves, are well described in the literature. The outer connective tissues, known as the epineurium and perineurium, include a double layer of collagen fibers that form a double helical wrapping that is visible with polarized light microscopy and by other contrast methods. This wrapping is a diagnostic characteristic of nerve tissues. Decades of research into dinosaur bones have produced interesting endocasts of probable brain morphology, but no soft and flexible nerve tissues have been reported from dinosaur remains. In this study we provide evidence of nerve fragments, characterized by a double helical wrapping of collagen fibers, from a Triceratops condyle collected at the Hell Creek Formation, MT. Based on comparison with nerves from an avian model we conclude that these are fragments of nerves that once resided in vivo in Triceratops.

Cryo-electron tomography (cryo-ET) has the potential to revolutionize our understanding of the building blocks of life since it provides the unique opportunity to study molecules and membrane architectures in the context of cellular interaction. In particular, the combination of fluorescence imaging with focused ion beam (FIB) milling allows the targeting of specific structures in thick cellular samples by preparing thin lamellae that contain a specific fluorescence marker. This technique has conventionally been time-consuming, as it requires sample transfer to multiple microscopes and presents several technical challenges that currently limit its success. Here we describe METEOR, a FIB-integrated microscopy solution that streamlines the correlative cryo-ET workflow. It protects the sample from ice contamination by minimizing handling steps, thus increasing the likelihood of high-quality data. It also allows for monitoring of the milling procedure to ensure the molecule of interest is captured and can then be imaged by cryo-ET.

The Royal Tyrrell Museum in Alberta, Canada is focused on reconstructing the communities that existed at the end of the Cretaceous. This challenge is made more difficult because for many of the taxa present, few complete specimens are preserved. In most smaller animals, all that remains preserved are isolated elements such as jaws. Teleost fish are of particular interest because they are dominant in today's aquatic communities. They are thought to have diversified after the Cretacious-Paleogene (K-Pg) mass extinction event, but many questions about early freshwater members of this group remain. Even the number of species present remains a mystery. Through an ambitious SR-μCT study we are non-destructively digitally dissecting both isolated elements and articulated skeletons that remain embedded in their rock matrix. Through comparison studies we are identifying features to aid in their identification while preserving the original fossils in an unaltered state for future studies.

In this work, we investigated the labeling efficiency of hydrophilic (DAPI) and hydrophobic (Nile Red) fluorescent probes using uni- and bi-directional diffusion on porcine liver tissue sections on the order of a few hundred microns in thickness. Bi-directional diffusion significantly enhanced the penetration of probes. Although diffusion most accurately describes the labeling process, we found that after removal of the labeling molecules residual probe molecules in the tissue sections continued to be transported deeper into the tissue resulting in additional tissue constituents being labeled. Our study shows that bi-directional labeling can significantly enhance the labeling of thick tissues for applications in which structural information in three dimensions is needed.

The rapid progress in the performance of today's sophisticated transmission electron microscopes also demands computational and educational tools capable of simulating the intricacy of electron image formation. The tools ideally should be widely accessible to the microscopy community and capable of covering both the breadth and depth demanded by modern materials science. Here, a cloud-based microscopy simulation platform, called cloudEMAPS and powered by cloud computing and modern server-client web programming architecture, is described. Compared to the current desktop solutions for electron microscopy simulations, cloud computing offers the unique advantages of on-demand, data sharing, high-performance computation and internet easy access using the state-of-the-art web infrastructure. This article will demonstrate these advantages using examples of interactive simulations of electron diffraction patterns and aberration-corrected electron optics.

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.