P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at “proof of concept” level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin–Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose–response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

It is difficult to believe that I have now completed five years as the Editor-in-Chief (EIC) of Microscopy and Microanalysis (MAM) and that this issue, Volume 20(1), is the start of the 20th year of the journal. Time passes quickly when one enjoys their work, and I have certainly enjoyed interacting with the majority of the many authors, reviewers, and editors associated with MAM. During my five years as EIC, we have seen significant growth of the journal and have experienced some growing pains along the way. Despite some of these growing pains and associated problems, I believe (although obviously a biased opinion) that we have been able to significantly enhance the quality of the manuscripts we are publishing and the overall product that the members of the Microscopy Society of America (MSA) and other subscribers receive. The purpose of this editorial is to provide some details on how much MAM has grown, to describe some of the problems and solutions associated with the growth, and to provide information to prospective authors on how to improve their experience of publishing in MAM by discussing some common errors that occur during submission and review of manuscripts.

This paper presents a statistical method to analyze instabilities that can be introduced during imaging in scanning electron microscopy (SEM). The method is based on the correlation of digital images and it can be used at different length scales. It consists of the evaluation of three different approaches with four parameters in total. The methodology is exemplified with a specific case of internal stress measurements where ion milling and SEM imaging are combined with digital image correlation. It is concluded that before these measurements it is important to test the SEM column to ensure the minimization and randomization of the imaging instabilities. The method has been applied onto three different field emission gun SEMs (Philips XL30, Tescan Lyra, FEI Helios 650) that represent three successive generations of SEMs. Important to note that the imaging instability can be quantified and its source can be identified.

Filament condition during hot-wire chemical vapor deposition conditions of multi-walled carbon nanotubes is a major concern for a stable deposition process. We report on the novel application of electron backscatter diffraction to characterize the carburization of tungsten filaments. During the synthesis, the W-filaments transform to W2C and WC. W-carbide growth followed a parabolic behavior corresponding to the diffusion of C as the rate-determining step. The grain size of W, W2C, and WC increases with longer exposure time and increasing filament temperature. The grain size of the recrystallizing W-core and W2C phase grows from the perimeter inwardly and this phenomenon is enhanced at filament temperatures in excess of 1,400°C. Cracks appear at filament temperatures >1,600°C, accompanied by a reduction in the filament operational lifetime. The increase of the W2C and recrystallized W-core grain size from the perimeter inwardly is ascribed to a thermal gradient within the filament, which in turn influences the hardness measurements and crack formation.

Attempts to elucidate chromosome structure have long remained elusive. Electron microscopy is useful for chromosome structure research because of its high resolution and magnification. However, biological samples such as chromosomes need to be subjected to various preparation steps, including dehydration, drying, and metal/carbon coating, which may induce shrinkage and artifacts. The ionic liquid technique has recently been developed and it enables sample preparation without dehydration, drying, or coating, providing a sample that is closer to the native condition. Concurrently, focused ion beam/scanning electron microscopy (FIB/SEM) has been developed, allowing the investigation and direct analysis of chromosome interiors. In this study, we investigated chromosome interiors by FIB/SEM using plant and human chromosomes prepared by the ionic liquid technique. As a result, two types of chromosomes, with and without cavities, were visualized, both for barley and human chromosomes prepared by critical point drying. However, chromosome interiors were revealed only as a solid structure, lacking cavities, when prepared by the ionic liquid technique. Our results suggest that the existence and size of cavities depend on the preparation procedures. We conclude that combination of the ionic liquid technique and FIB/SEM is a powerful tool for chromosome study.

Two related aspects of nano-droplet condensation and droplets coalescence are studied for droplets on self-supported thin water films. The experiments are conducted in the environmental scanning electron microscope using wet scanning transmission electron microscopy. Favorable condensation sites are examined and in-situ position-controlled condensation experiments are conducted. The interaction among condensed multi-droplets as well as between a single droplet and the underneath nano-thick water film are dynamically examined with 10nm lateral resolution. The droplet round shape is reshaped to flat-like facets in-between droplets of 30-230 nm separation. Dynamic imaging of a few minutes duration shows a delayed coalescence effect, being explained by increased droplet-droplet electrostatic interaction relative to van der Waals interaction.

We report a new simple but effective method to extract the weak surface signals from a “thickness series” of recorded electron energy-loss spectra. Using precise thickness measurements and energy-loss near-edge structures measured at increasing thicknesses, we are able to extract the surface and bulk components in the series. The electronic structure and bonding information from SrTiO3 (001) reconstructed surfaces have been successfully obtained by applying this approach. This approach can be applied to study many other cases including absorbed monolayers and beam-sensitive interfaces.

A procedure based on focused ion beam milling and in situ lift-out is introduced for the preparation of high-quality specimens for in situ annealing experiments in the transmission electron microscope. The procedure allows an electron-transparent lamella to be cleaned directly on a heating chip using a low ion energy and back-side milling in order to minimize redeposition and damage. The approach is illustrated through the preparation of an Al–Mn–Fe complex metallic alloy specimen.

Biofilms are three-dimensional communities of bacteria distributed in a highly hydrated extracellular matrix (ECM). They can be visualized by scanning electron microscopy (SEM), but the requisite SEM sample preparation can modify the biofilm morphology. Here, four different approaches to prepare biofilms of hydrated Staphylococcus aureus for SEM imaging are compared. In order of increasing cooling effectiveness these are: (1) drying in air; (2) plunging in liquid nitrogen; (3) plunging in liquid ethane; and (4) high pressure freezing with liquid nitrogen. These different methods give rise to markedly different biofilm morphologies, which are revealed by cryo-SEM imaging. Significantly, high-pressure frozen biofilms exhibit a rich network of nanoscale ECM fibers surrounding individual bacteria throughout the biofilm thickness. This structure is entirely lost when similar biofilms are dried in air, and it is substantially modified when these biofilms are plunged into liquid nitrogen or liquid ethane.

Taking advantage of previous measurements by Geiger and co-workers, we discuss the possibilities and problems of measuring vibrational modes of energy loss in a transmission electron microscope fitted with a monochromator and a high-resolution energy-loss spectrometer. The tail of the zero-loss peak is seen to be a major limitation, rather than its full-width at half-maximum. Because of the low oscillator strengths and small cross-sections involved, radiation damage will limit the spatial resolution if this technique is applied to organic specimens. Delocalization of the inelastic scattering may also be a limitation, if a dipole description of the scattering process is valid.