Single-molecule localization microscopy (SMLM) is a family of super-resolution microscopy techniques based on localizing clusters of detected photons that are emitted by single molecules. The localization procedure is based on careful statistical analysis of long image sequences to derive the nanometer positions of the molecules. By introducing additional optics, such as cylindrical lenses in the optical system, SMLM techniques have been extended to 3D super-resolution imaging. This adds a calibration step, thereby further complicating the data analysis. Here we present Huygens Localizer, a well-supported user-friendly package that carries out these tasks quickly by offloading carefully designed 2D and 3D analysis and visualization procedures to massively parallel graphical processors (GPUs).

This is the first of two articles on the Extant Life Volumetric Imaging System (ELVIS) describing a combined digital holographic microscope (DHM) and a fluorescence light-field microscope (FLFM). The instrument is modular and robust enough for field use. Each mode uses its own illumination source and camera, but both microscopes share a common objective lens and sample viewing chamber. This allows correlative volumetric imaging in amplitude, quantitative phase, and fluorescence modes. A detailed schematic and parts list is presented, as well as links to open-source software packages for data acquisition and analysis that permits interested researchers to duplicate the design. Instrument performance is quantified using test targets and beads. In the second article on ELVIS, to be published in the next issue of Microscopy Today, analysis of data from field tests and images of microorganisms will be presented.

Researchers in the Physics Department of St. Olaf College are using a uniquely designed, integrated nanoindenter-quartz microbalance apparatus to bridge the gap between the fundamental science of friction and the engineering of practical micromechanical systems. This level of micro-research requires extreme stability for the microbalance instrumentation. Since 2001, the lab has used negative-stiffness vibration isolation to achieve a high level of isolation in multiple directions, custom tailoring resonant frequencies to 0.5 Hz vertically and horizontally.

Following the previous article, here we describe the first field demonstration of the ELVIS system, performed at Newport Beach, CA. We examined ocean water to detect microorganisms using the combined holographic and light-field fluorescence microscope and successfully detected both eukaryotes and prokaryotes. The shared field of view provided simultaneous bright-field (amplitude), phase, and fluorescence information from both chlorophyll autofluorescence and acridine orange staining. The entire process was performed in a nearly autonomous manner using a specifically designed sample processing unit (SPU) and custom acquisition software. We also discuss improvements to the system made after the field test that will make it more broadly useful to other types of fluorophores and samples.

Astronauts who spend significant time in space may experience intracranial hypertension (pressure inside the skull), which often does not normalize upon return to Earth. This type of intracranial pressure can cause vision problems, headaches, glaucoma, and other serious health problems, making it a significant obstacle to long-duration space exploration missions. NASA is hoping that a better understanding of how central nervous system stem cells divide in microgravity will help lead to ways to protect astronauts from problems with intracranial pressure and to design adequate preventive measures. These studies examine cell growth and division of neural stem cells that experienced microgravity on the International Space Station and were returned to normal gravity prior to live-cell imaging studies to determine cell changes.

The spatial organization of chromatin has become recognized as a major factor in the regulation of gene expression. While chromosome conformation capture techniques, such as high-throughput chromosome conformation capture (Hi-C), have advanced our understanding of the role of 3D organization of chromatin on chromosomal function, Hi-C data are typically ensemble in nature and relies on intense computational methods to derive genomic structural information. The field is beginning to shift in recognizing the potential value of imaging methods to study spatial organization of chromatin. Imaging allows acquisition and analysis at the individual cell level while providing direct visualization and analysis of 3D chromatin organization. This feature will highlight how single-molecule localization microscopy (SMLM) using the Bruker Vutara SMLM system in conjunction with sequential labeling via microfluidics is revolutionizing our understanding of nuclear spatial organization and its role in regulation of gene function.

There is often a need to locate the same cellular structure of interest in light and electron microscopy, which can be a difficult task. Here we present a method that uses only commercially available reagents and standard epi-fluorescence and transmission electron microscopy (TEM) technology to make correlative light and electron microscopy (CLEM) available to a large group of researchers without specialized CLEM hardware. This was achieved by seeding cells on photo-etched gridded cover slips and staining the protein to be localized with a secondary antibody coupled to both a fluorophore and 10 nm gold. The presence of the grid allowed for the alignment of light microscopy images with TEM images and the double-labeled antibody revealed co-localization of the fluorophore with gold particles.

Despite its vital role in advancing live cell imaging (LCI) and unraveling the complex processes that control cellular function, fluorescence microscopy presents challenges for the researcher, including labeling agents that can interfere with normal molecular activities, and limitations on repeat measurements and long-term studies from photobleaching and phototoxicity phenomena. Holotomography microscopy delivers nanoscale, label-free, real-time LCI and can combine this quantitative phase imaging (QPI) with fluorescence for state-of-the-art spatiotemporal resolution as well as high molecular specificity. This article introduces QPI, examines its advantages for LCI, and reviews correlative microscopy studies in cell pathophysiology in combination with fluorescence.

A low-cost infrared microscope is evaluated in both transmission and attenuated total internal reflection (ATR) sampling modes. Transmission spectra of 60 to 250 micrometer samples exhibited an excellent signal-to-noise ratio. Signal-to-noise in transmission mode also correlated well with theoretical predictions. The quantitative capability of the microscope was demonstrated by measuring the colorant concentration of red, yellow, and blue polypropylene fibers. These latter measurements were conducted using the ATR sampling mode of the microscope. Colorant concentrations could be measured to 1% by weight, and calibration curves with excellent R2 values were obtained over the dye concentration range of 1 to 10% by weight colorant.

Optical Photothermal Infrared (O-PTIR) spectroscopy is a new technique for measuring submicron spatial resolution IR spectra with little or no sample preparation. This speeds up analysis times benefiting high-volume manufacturers through gaining insight into process contamination that occurs during development and on production lines. The ability to rapidly obtain far-field non-contact IR spectra at high spatial resolution facilitates the chemical identification of small organic contaminants that are not possible to measure with conventional Fourier transform infrared (FT-IR) microspectroscopy. The unique pump-probe system architecture also facilitates submicron simultaneous IR + Raman microscopy from the same spot with the same spatial resolution. With these unique capabilities, O-PTIR is finding utilization in the high-volume and high-value industries of high-tech componentry (memory storage, electronics, displays, etc.).

The Cyanidiophyceae are a class of unicellular red algae that live in environments at the extremes of temperature, pH, salt, and heavy metal concentrations. These photosynthetic organisms have been able to occupy these rather unusual niches by acquiring a variety of genes from extremophilic prokaryotes and/or viruses via the process of horizontal gene transfer (HGT). Elucidating how the proteins encoded by these genes integrate into existing metabolic systems is crucial to understanding and evaluating the significance of HGT in eukaryotes and its role in conferring adaptive traits. This article addresses the fundamentals of HGT in model red algae, its function as a driver of evolution across the web of life with a focus on eukaryotes, and summarizes ongoing work and the future directions of this field.

Investigation of the Fezouata Shale has added to our knowledge on the initial diversification of metazoans. These Lower Ordovician deposits yielded abundant and diverse remains of cuticularized to lightly sclerotized organisms, in addition to numerous soft tissues. Described fossilized soft parts recovered from the Fezouata Shale belong mainly to arthropods. Soft tissues in echinoderms, a main component of the Fezouata Biota, remain largely unexplored. Here, we show that soft tissue-like impressions previously reported in eocrinoid and somasteroid echinoderms from this formation, are the results of modern weathering and fossil preparation that involved the use of latex molds: they do not reflect any original (soft) anatomical features of these organisms. These two examples suggest that reports of putative soft parts, especially in taxa with no current representatives, need to be thoroughly and critically evaluated.

Two standardless quantitative methods for evaluating EDS X-ray spectra were investigated in regards their basic metrics. Both methods have similar total errors, but the error contributions are from different sources. In the P/B-based method, error is more related to counting statistics and therefore can benefit from high count rates achievable with modern silicon drift detectors. To reduce systematic uncertainties in the net-count-based standardless approach, measured values need to be supported by data in a previously measured database. Using the P/B-based method, it is now possible to achieve standardless EDS quantification within ±10% relative deviation from true composition for 95% of results.

Based on technologies capable of data collection between the millimeter and nanometer scales, correlative imaging has been transforming how researchers obtain molecular and spatial information from specimens. Attempts to combine multidimensional data are often met with the challenge of overcoming suboptimal sample conditions such as reduced fluorescence signal, poor specimen preservation, anisotropic specimen deformation, and low specimen contrast. These issues motivated the development and use of enhanced sample preparation procedures, as well as specialized imaging software to overcome such challenges. In this work we present three simple methods to correlate optical and scanning electron microscopy images.