X-ray micro-computed tomography (μCT) is commonly used for imaging of samples in biomedical or materials science research. Owing to the ability to visualize a sample in a nondestructive way, X-ray μCT is perfectly suited to inspect fossilized specimens, which are mostly unique or rare. In certain regions of the world where important sedimentation events occurred in the Precambrian geological time, several fossilized animals are studied to understand questions related to their origin, environment, and life evolution. This article demonstrates the advantages of applying absorption and phase-contrast CT on the enigmatic fossil Corumbella werneri, one of the oldest known animals capable of building hard parts, originally discovered in Corumbá (Brazil). Different tomographic setups were tested to visualize the fossilized inner structures: a commercial laboratory-based μCT device, two synchrotron-based imaging setups using conventional absorption and propagation-based phase contrast, and a commercial X-ray microscope with a lens-coupled detector system, dedicated for radiography and tomography. Based on our results we discuss the strengths and weaknesses of the different imaging setups for paleontological studies.

A simple model is proposed to account for the loss of collected X-ray signal by the shadowing of X-ray detectors in the scanning transmission electron microscope. The model is intended to aid the analysis of three-dimensional elemental data sets acquired using energy-dispersive X-ray tomography methods where shadow-free specimen holders are unsuitable or unavailable. The model also provides a useful measure of the detection system geometry.

We conducted a comparative study of three widely used algorithms for the detection of fiducial markers in electron microscopy images. The algorithms were applied to four datasets from different sources. For the purpose of obtaining comparable results, we introduced figures of merit and implemented all three algorithms in a unified code base to exclude software-specific differences. The application of the algorithms revealed that none of the three algorithms is superior to the others in all cases. This leads to the conclusion that the choice of a marker detection algorithm highly depends on the properties of the dataset to be analyzed, even within the narrowed domain of electron tomography.

We describe a system for rapidly screening hundreds of nanoparticle samples using transmission electron microscopy (TEM). The system uses a liquid handling robot to place up to 96 individual samples onto a single standard TEM grid at separate locations. The grid is then transferred into the TEM and automated software is used to acquire multiscale images of each sample. The images are then analyzed to extract metrics on the size, shape, and morphology of the nanoparticles. The system has been used to characterize plasmonically active nanomaterials.

The present work investigates the applicability of nonlinear imaging microscopy for the precise assessment of degradation of the outer protective layers of painted artworks as a function of depth due to aging. Two fresh and artificially aged triterpenoid varnishes, dammar and mastic, were tested. Nonlinear imaging techniques have been employed as a new diagnostic tool for determination of the exact thickness of the affected region due to artificial aging of the natural varnishes. The measured thicknesses differ from the calculated mean penetration depths of the samples. These nondestructive, high resolution modalities are valuable analytical tools for aging studies and they have the potential to provide unique in-depth information. Single photon laser induced fluorescence measurements and Raman spectroscopy were used for the integrated investigation and analysis of aging effects in varnishes.

Frangula azorica V. Grubow is a Macaronesian flora medicinal plant, endemic from Azores islands and inscribed on the International Union for the Conservation of Nature (IUCN) Red List of Threatened Species. This species, known as “sanguinho,” belongs to the family Rhamnaceae, the same as Frangula alnus Mill. and Frangula purshiana (DC.) J. G. Cooper, two widely used official laxative herbal medicines of the western Pharmacopoeias constituted by the dried barks of each species. Morphological and chemical studies on F. azorica dried bark are scarce although it is potentially recognized as a Portuguese laxative herbal medicine. Macroscopically, the bark occurs in quills or nearly flat pieces. A channeled external surface with transversely elongated lenticels is characteristic. When the outer phellem layer is removed, a bright purple inner phellem layer is disclosed. Light and electron microscopy observations revealed flattened phellem cells with slightly thickened walls, cortical parenchyma with secretory ducts and groups of sclereids, phloem with groups of fibers and sheaths of parenchymatous cells containing druses or more frequently prismatic crystals of calcium oxalate, and parenchymatous medullary rays one to three cells wide with spherical starch grains. Observation of these botanical characteristics must be included in quality monographs of F. azorica bark herbal medicine.

Scanning electron microscopy (SEM) of nanoscale objects in dry and fully hydrated conditions at different temperatures is of critical importance in revealing details of their interactions with an ambient environment. Currently available WETSEM capsules are equipped with thin electron-transparent membranes and allow imaging of samples at atmospheric pressure, but do not provide temperature control over the sample. Here, we developed and tested a thermoelectric cooling/heating setup for WETSEM capsules to allow ambient pressure in situ SEM studies with a temperature range between −15 and 100°C in gaseous, liquid, and frozen conditions. The design of the setup also allows for correlation of the SEM with optical microscopy and spectroscopy. As a demonstration of the possibilities of the developed approach, we performed real-time in situ microscopy studies of water condensation on a surface of Morpho sulkowskyi butterfly wing scales. We observed that initial water nucleation takes place on top of the scale ridges. These results confirmed earlier discovery of a preexisting polarity gradient of the ridges of Morpho butterflies. Our developed thermoelectric cooling/heating setup for environmental capsules meets the diverse needs for in situ nanocharacterization in material science, catalysis, microelectronics, chemistry, and biology.

Optical projection tomography (OPT) is a computed tomography technique at optical frequencies for samples of 0.5–15 mm in size, which fills an important “imaging gap” between confocal microscopy (for smaller samples) and large-sample methods such as fluorescence molecular tomography or micro magnetic resonance imaging. OPT operates in either fluorescence or transmission mode. Two-dimensional (2D) projections are taken over 360° with a fixed rotational increment around the vertical axis. Standard 3D reconstruction from 2D OPT uses the filtered backprojection (FBP) algorithm based on the Radon transform. FBP approximates the inverse Radon transform using a ramp filter that spreads reconstructed pixels to neighbor pixels thus producing streak and other types of artifacts, as well as noise. Artifacts increase the variation of grayscale values in the reconstructed images. We present an algorithm that improves the quality of reconstruction even for a low number of projections by simultaneously minimizing the sum of absolute brightness changes in the reconstructed volume (the total variation) and the error between measured and reconstructed data. We demonstrate the efficiency of the method on real biological data acquired on a dedicated OPT device.

Whole slide imaging (WSI) can be used to quantify multiple responses within tissue sections during histological analysis. Feature Analysis on Consecutive Tissue Sections (FACTS®) allows the investigator to perform digital morphometric analysis (DMA) within specified regions of interest (ROI) across multiple serial sections at faster rates when compared with manual morphometry methods. Using FACTS® in conjunction with WSI is a powerful analysis tool, which allows DMA to target specific ROI across multiple tissue sections stained for different biomarkers. DMA may serve as an appropriate alternative to classic, manual, histologic morphometric measures, which have historically relied on the selection of high-powered fields of views and manual scoring (e.g., a gold standard). In the current study, existing preserved samples were used to determine if DMA would provide similar results to manual counting methods. Rodent hearts (n=14, left ventricles) were stained with Masson’s trichrome, and reacted for cluster of differentiation 68 (CD-68). This study found no statistical significant difference between a classic, manual method and the use of digital algorithms to perform the similar counts (p=0.38). DMA offers researchers the ability to accurately evaluate morphological characteristics in a reproducible fashion without investigator bias and with higher throughput.