Magnetotactic bacteria: concepts, conundrums, and insights from a novel in situ approach using digital holographic microscopy (DHM).

Magnetotactic bacteria (MTB) are a diverse group of highly motile Gram-negative microorganisms with the common ability to orient along magnetic field lines, a behavior known as magnetotaxis. Ubiquitous in aquatic sediment environments, MTB are often microaerophilic and abundant at the oxic/anoxic interface. Magnetic field sensing is accomplished using intracellular, membrane-encased, iron-containing minerals known as magnetosomes. The chemistry, morphology and arrangement of magnetosomes differs substantially among different MTB. Although magnetic field sensing mechanisms, genetic bases and protein functions have been elucidated in select model organisms such as the Magnetospirillum strains and Desulfovibrio RS-1, not all findings are applicable to diverse clades of MTB. As the number of identified species has increased, it has become evident that many of the characteristics and mechanisms once presumed to be prototypical of MTB are in fact not universal. Here we present a general overview of the current state of MTB research for readers outside of the realm of prokaryotic research, focusing on recent discoveries, knowledge gaps and future directions. In addition, we report new insights acquired using holographic technology to observe and quantify microbial responses in magnetic fields that are earth-strength or weaker, providing a new ecophysiological approach to in situ MTB research.

Using the Gouy phase anomaly to localize and track bacteria in digital holographic microscopy 4D images.

Described over 100 years ago, the Gouy phase anomaly refers to the additional π phase shift that is accumulated as a wave passes through focus. It is potentially useful in analyzing any type of phase-sensitive imaging; in light microscopy, digital holographic microscopy (DHM) provides phase information in the encoded hologram. One limitation of DHM is the weak contrast generated by many biological cells, especially unpigmented bacteria. We demonstrate here that the Gouy phase anomaly may be detected directly in the phase image using the z-derivative of the phase, allowing for precise localization of unlabeled, micrometer-sized bacteria. The use of dyes that increase phase contrast does not improve detectability. This approach is less computationally intensive than other procedures such as deconvolution and is relatively insensitive to reconstruction parameters. The software is implemented in an open-source FIJI plug-in.

Genetically Encoded Phase Contrast Agents for Digital Holographic Microscopy.

Quantitative phase imaging and digital holographic microscopy have shown great promise for visualizing the motion, structure, and physiology of microorganisms and mammalian cells in three dimensions. However, these imaging techniques currently lack molecular contrast agents analogous to the fluorescent dyes and proteins that have revolutionized fluorescence microscopy. Here we introduce the first genetically encodable phase contrast agents based on gas vesicles. The relatively low index of refraction of the air-filled core of gas vesicles results in optical phase advancement relative to aqueous media, making them a "positive" phase contrast agent easily distinguished from organelles, dyes, or microminerals. We demonstrate this capability by identifying and tracking the motion of gas vesicles and gas vesicle-expressing bacteria using digital holographic microscopy, and by imaging the uptake of engineered gas vesicles by mammalian cells. These results give phase imaging a biomolecular contrast agent, expanding the capabilities of this powerful technology for three-dimensional biological imaging.

Enhancing final image contrast in off-axis digital holography using residual fringes.

We show that background fringe-pattern subtraction is a useful technique for removing static noise from off-axis holographic reconstructions and can enhance image contrast in volumetric reconstructions by an order of magnitude in the case for instruments with relatively stable fringes. We demonstrate the fundamental principle of this technique and introduce some practical considerations that must be made when implementing this scheme, such as quantifying fringe stability. This work also shows an experimental verification of the background fringe subtraction scheme using various biological samples.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM).

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Digital Holographic Microscopy, a Method for Detection of Microorganisms in Plume Samples from Enceladus and Other Icy Worlds.

Detection of extant microbial life on Earth and elsewhere in the Solar System requires the ability to identify and enumerate micrometer-scale, essentially featureless cells. On Earth, bacteria are usually enumerated by culture plating or epifluorescence microscopy. Culture plates require long incubation times and can only count culturable strains, and epifluorescence microscopy requires extensive staining and concentration of the sample and instrumentation that is not readily miniaturized for space. Digital holographic microscopy (DHM) represents an alternative technique with no moving parts and higher throughput than traditional microscopy, making it potentially useful in space for detection of extant microorganisms provided that sufficient numbers of cells can be collected. Because sample collection is expected to be the limiting factor for space missions, especially to outer planets, it is important to quantify the limits of detection of any proposed technique for extant life detection. Here we use both laboratory and field samples to measure the limits of detection of an off-axis digital holographic microscope (DHM). A statistical model is used to estimate any instrument's probability of detection at various bacterial concentrations based on the optical performance characteristics of the instrument, as well as estimate the confidence interval of detection. This statistical model agrees well with the limit of detection of 103 cells/mL that was found experimentally with laboratory samples. In environmental samples, active cells were immediately evident at concentrations of 104 cells/mL. Published estimates of cell densities for Enceladus plumes yield up to 104 cells/mL, which are well within the off-axis DHM's limits of detection to confidence intervals greater than or equal to 95%, assuming sufficient sample volumes can be collected. The quantitative phase imaging provided by DHM allowed minerals to be distinguished from cells. Off-axis DHM's ability for rapid low-level bacterial detection and counting shows its viability as a technique for detection of extant microbial life provided that the cells can be captured intact and delivered to the sample chamber in a sufficient volume of liquid for imaging. Key Words: In situ life detection-Extant microorganisms-Holographic microscopy-Ocean Worlds-Enceladus-Imaging. Astrobiology 17, 913-925.

A Submersible, Off-Axis Holographic Microscope for Detection of Microbial Motility and Morphology in Aqueous and Icy Environments.

Sea ice is an analog environment for several of astrobiology's near-term targets: Mars, Europa, Enceladus, and perhaps other Jovian or Saturnian moons. Microorganisms, both eukaryotic and prokaryotic, remain active within brine channels inside the ice, making it unnecessary to penetrate through to liquid water below in order to detect life. We have developed a submersible digital holographic microscope (DHM) that is capable of resolving individual bacterial cells, and demonstrated its utility for immediately imaging samples taken directly from sea ice at several locations near Nuuk, Greenland. In all samples, the appearance and motility of eukaryotes were conclusive signs of life. The appearance of prokaryotic cells alone was not sufficient to confirm life, but when prokaryotic motility occurred, it was rapid and conclusive. Warming the samples to above-freezing temperatures or supplementing with serine increased the number of motile cells and the speed of motility; supplementing with serine also stimulated chemotaxis. These results show that DHM is a useful technique for detection of active organisms in extreme environments, and that motility may be used as a biosignature in the liquid brines that persist in ice. These findings have important implications for the design of missions to icy environments and suggest ways in which DHM imaging may be integrated with chemical life-detection suites in order to create more conclusive life detection packages.