Holographic microscopy for 3D tracking of bacteria

Understanding when, how, and if bacteria swim is key to understanding critical ecological and biological processes, from carbon cycling to infection. Imaging motility by traditional light microscopy is limited by focus depth, requiring cells to be constrained in z. Holographic microscopy offers an instantaneous 3D snapshot of a large sample volume, and is therefore ideal in principle for quantifying unconstrained bacterial motility. However, resolving and tracking individual cells is difficult due to the low amplitude and phase contrast of the cells; the index of refraction of typical bacteria differs from that of water only at the second decimal place. In this work we present a combination of optical and sample-handling approaches to facilitating bacterial tracking by holographic phase imaging. The first is the design of the microscope, which is an off-axis design with the optics along a common path, which minimizes alignment issues while providing all of the advantages of off-axis holography. Second, we use anti-reflective coated etalon glass in the design of sample chambers, which reduce internal reflections. Improvement seen with the antireflective coating is seen primarily in phase imaging, and its quantification is presented here. Finally, dyes may be used to increase phase contrast according to the Kramers-Kronig relations. Results using three test strains are presented, illustrating the different types of bacterial motility characterized by an enteric organism (Escherichia coli), an environmental organism (Bacillus subtilis), and a marine organism (Vibrio alginolyticus). Data processing steps to increase the quality of the phase images and facilitate tracking are also discussed

Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype

Robust, compact implementation of an off-axis digital holographic microscope

Recent advances in digital technologies, such as high-speed computers and large-format digital imagers, have led to a burgeoning interest in the science and engineering of digital holographic microscopy (DHM). Here we report on a novel off-axis DHM, based on a twin-beam optical design, which avoids the limitations of prior systems, and provides many advantages, including compactness, intrinsic stability, robustness against misalignment, ease of use, and cost. These advantages are traded for a physically constrained sample volume, as well as a fixed fringe spacing. The first trade is not overly restrictive for most applications, and the latter provides for a pre-set assembly alignment that optimizes the spatial frequency sampling. Moreover, our new design supports use in both routine laboratory settings as well as extreme environments without any sacrifice in performance, enabling ready observation of microbial species in the field. The instrument design is presented in detail here, along with a demonstration of bacterial video imaging at sub-micrometer resolution at temperatures down to –15 °C

Volatile Organic Compounds and Pulmonary Function in the Third National Health and Nutrition Examination Survey, 1988–1994

BACKGROUND: Volatile organic compounds (VOCs) are present in much higher concentrations indoors, where people spend most of their time, than outdoors and may have adverse health effects. VOCs have been associated with respiratory symptoms, but few studies address objective respiratory end points such as pulmonary function. Blood levels of VOCs may be more indicative of personal exposures than are air concentrations; no studies have addressed their relationship with respiratory outcomes. OBJECTIVE: We examined whether concentrations of 11 VOCs that were commonly identified in blood from a sample of the U.S. population were associated with pulmonary function. METHODS: We used data from 953 adult participants (20–59 years of age) in the Third National Health and Nutrition Examination Survey (1988–1994) who had VOC blood measures as well as pulmonary function measures. Linear regression models were used to evaluate the relationship between 11 VOCs and measures of pulmonary function. RESULTS: After adjustment for smoking, only 1,4-dichlorobenzene (1,4-DCB) was associated with reduced pulmonary function. Participants in the highest decile of 1,4-DCB concentration had decrements of −153 mL [95% confidence interval (CI), −297 to −8] in forced expiratory volume in 1 sec and −346 mL/sec (95% CI, −667 to −24) in maximum mid-expiratory flow rate, compared with participants in the lowest decile. CONCLUSIONS: Exposure to 1,4-DCB, a VOC related to the use of air fresheners, toilet bowl deodorants, and mothballs, at levels found in the U.S. general population, may result in reduced pulmonary function. This common exposure may have long-term adverse effects on respiratory health

A Multi-Modal Volumetric Microscope with Automated Sample Handling for Surveying Microbial Life in Liquid Samples

In the study of microbial life, microscopy plays a unique role due to its ability to detect ordered structure, motility, and fluorescence signals. As such it has also recently gained attention in the context of searching for extant life on distant Solar System bodies bearing liquid water. In this paper we introduce a multimodal volumetric microscopy system for potential future spaceflight missions that combines digital holographic microscopy (DHM) and volume fluorescence imager (VFI), which are volumetric imaging methods that provide highresolution, high-throughput examination of liquid samples. DHM provides information on the absorption, morphology, and motility of imaged objects without requiring the use of contrast agents. On the other hand, VFI based on light field microscopy focuses on the fluorescence signals from the sample to observe specific structures dyed with targeted contrast agents or providing unique autofluorescence signals. We also present an autonomous sample handling and data acquisition system to allow for an autonomous mission to distant planets or moons, or for autonomous use in bodies of water on Earth. The full system, named ELVIS, or Extant Life Volumetric Imaging System, is capable of autonomously surveying a liquid sample to extract morphology, motility, and fluorescence signals of extant microbial life

ELVIS: A Correlated Light-Field and Digital Holographic Microscope for Field and Laboratory Investigations

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

Subglacial Meltwater Supported Aerobic Marine Habitats During Snowball Earth

The Earth’s most severe ice ages interrupted a crucial interval in eukaryotic evolution with widespread ice coverage during the Cryogenian Period (720 to 635 Ma). Aerobic eukaryotes must have survived the “Snowball Earth” glaciations, requiring the persistence of oxygenated marine habitats, yet evidence for these environments is lacking. We examine iron formations within globally distributed Cryogenian glacial successions to reconstruct the redox state of the synglacial oceans. Iron isotope ratios and cerium anomalies from a range of glaciomarine environments reveal pervasive anoxia in the ice-covered oceans but increasing oxidation with proximity to the ice shelf grounding line. We propose that the outwash of subglacial meltwater supplied oxygen to the synglacial oceans, creating glaciomarine oxygen oases. The confluence of oxygen-rich meltwater and iron-rich seawater may have provided sufficient energy to sustain chemosynthetic communities. These processes could have supplied the requisite oxygen and organic carbon source for the survival of early animals and other eukaryotic heterotrophs through these extreme glaciations

Choroid Development and Feasibility of Choroidal Imaging in the Preterm and Term Infants Utilizing SD-OCT

To determine whether choroidal imaging is feasible in preterm and term infants using an 840-nm portable spectral domain optical coherence tomography (SD-OCT) system without the use of enhanced-depth imaging techniques and to assess choroidal development by comparing choroidal thickness of preterm infants, term infants, and adults