Fluorescence Microscopy with Deep UV, Near UV, and Visible Excitation for In Situ Detection of Microorganisms.

We report a simple, inexpensive design of a fluorescence microscope with light-emitting diode (LED) excitation for detection of labeled and unlabeled microorganisms in mineral substrates. The use of deep UV (DUV) excitation with visible emission requires no specialized optics or slides and can be implemented easily and inexpensively using an oblique illumination geometry. DUV excitation (<280 nm) is preferable to near UV (365 nm) for avoidance of mineral autofluorescence. When excited with DUV, unpigmented bacteria show two emission peaks: one in the near UV ∼320 nm, corresponding to proteins, and another peak in the blue to green range, corresponding to flavins and/or reduced nicotinamide adenine dinucleotide (NADH). Many commonly used dyes also show secondary excitation peaks in the DUV, with identical emission spectra and quantum yields as their primary peak. However, DUV fails to excite key biosignature molecules, especially chlorophyll in cyanobacteria. Visible excitation (violet to blue) also results in less mineral autofluorescence than near UV, and most autofluorescence in the minerals seen here is green, so that red dyes and red autofluorescence of chlorophyll and porphyrins are readily distinguished. The pairing of DUV and near UV or visible excitation, with emission across the visible, represents the most thorough approach to detection of labeled and unlabeled bacteria in soil and rock.

Detectability of unresolved particles in off-axis digital holographic microscopy.

Off-axis digital holographic microscopy (DHM) provides both amplitude and phase images, and so it may be used for label-free 3D tracking of micro- and nano-sized particles of different compositions, including biological cells, strongly absorbing particles, and strongly scattering particles. Contrast is provided by differences in either the real or imaginary parts of the refractive index (phase contrast and absorption) and/or by scattering. While numerous studies have focused on phase contrast and improving resolution in DHM, particularly axial resolution, absent have been studies quantifying the limits of detection for unresolved particles. This limit has important implications for microbial detection, including in life-detection missions for space flight. Here we examine the limits of detection of nanosized particles as a function of particle optical properties, microscope optics (including camera well depth and substrate), and data processing techniques and find that DHM provides contrast in both amplitude and phase for unresolved spheres, in rough agreement with Mie theory scattering cross-sections. Amplitude reconstructions are more useful than phase for low-index spheres and should not be neglected in DHM analysis.

The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates.

The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800 nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM enabled discrimination of SYTO 9 labelled cells from background, but that the lifetime of SYTO 9 was shorter in cells on minerals than in pure culture under our conditions. Photosynthetic microorganisms were easily observed using both FLIM and confocal. Unlabelled, nonpigmented bacteria showed weak signals that were difficult to distinguish from background when minerals were present, though cellular autofluorescence consistent with NAD(P)H could be seen in pure cultures, and phasor analysis permitted detection on rocks. Gypsum and marble samples showed similar autofluorescence profiles, with little autofluorescence in the yellow-to-red range. Lifetime or time-gated imaging may prove a useful tool for environmental microbiology. LAY DESCRIPTION: The standard method of bacterial enumeration is to label the cells with a fluorescent dye and count them under high-power fluorescence microscopy. However, this can be difficult when the cells are embedded in soil and rock due to fluorescence from the surrounding minerals and dye binding to ambiguous features of the substrate. The use of fluorescence lifetime imaging (FLIM) can disambiguate these signals and allow for improved detection of bacteria in environmental samples.

Microbial Motility at the Bottom of North America: Digital Holographic Microscopy and Genomic Motility Signatures in Badwater Spring, Death Valley National Park.

Motility is widely distributed across the tree of life and can be recognized by microscopy regardless of phylogenetic affiliation, biochemical composition, or mechanism. Microscopy has thus been proposed as a potential tool for detection of biosignatures for extraterrestrial life; however, traditional light microscopy is poorly suited for this purpose, as it requires sample preparation, involves fragile moving parts, and has a limited volume of view. In this study, we deployed a field-portable digital holographic microscope (DHM) to explore microbial motility in Badwater Spring, a saline spring in Death Valley National Park, and complemented DHM imaging with 16S rRNA gene amplicon sequencing and shotgun metagenomics. The DHM identified diverse morphologies and distinguished run-reverse-flick and run-reverse types of flagellar motility. PICRUSt2- and literature-based predictions based on 16S rRNA gene amplicons were used to predict motility genotypes/phenotypes for 36.0-60.1% of identified taxa, with the predicted motile taxa being dominated by members of Burkholderiaceae and Spirochaetota. A shotgun metagenome confirmed the abundance of genes encoding flagellar motility, and a Ralstonia metagenome-assembled genome encoded a full flagellar gene cluster. This study demonstrates the potential of DHM for planetary life detection, presents the first microbial census of Badwater Spring and brine pool, and confirms the abundance of mobile microbial taxa in an extreme environment.

Recent advances in experimental design and data analysis to characterize prokaryotic motility.

Bacterial motility plays a key role in important cell processes such as chemotaxis and biofilm formation, but is challenging to quantify due to the small size of the individual microorganisms and the complex interplay of biological and physical factors that influence motility phenotypes. Swimming, the first type of motility described in bacteria, still remains largely unquantified. Light microscopy has enabled qualitative characterization of swimming patterns seen in different strains, such as run and tumble, run-reverse-flick, run and slow, stop and coil, and push and pull, which has allowed for elucidation of the underlying physics. However, quantifying these behaviors (e.g., identifying run distances and speeds, turn angles and behavior by surfaces or cell-cell interactions) remains a challenging task. A qualitative and quantitative understanding of bacterial motility is needed to bridge the gap between experimentation, omics analysis, and bacterial motility theory. In this review, we discuss the strengths and limitations of how phase contrast microscopy, fluorescence microscopy, and digital holographic microscopy have been used to quantify bacterial motility. Approaches to automated software analysis, including cell recognition, tracking, and track analysis, are also discussed with a view to providing a guide for experimenters to setting up the appropriate imaging and analysis system for their needs.

Quantification of Motility in Bacillus subtilis at Temperatures Up to 84°C Using a Submersible Volumetric Microscope and Automated Tracking.

We describe a system for high-temperature investigations of bacterial motility using a digital holographic microscope completely submerged in heated water. Temperatures above 90°C could be achieved, with a constant 5°C offset between the sample temperature and the surrounding water bath. Using this system, we observed active motility in Bacillus subtilis up to 66°C. As temperatures rose, most cells became immobilized on the surface, but a fraction of cells remained highly motile at distances of >100 µm above the surface. Suspended non-motile cells showed Brownian motion that scaled consistently with temperature and viscosity. A novel open-source automated tracking package was used to obtain 2D tracks of motile cells and quantify motility parameters, showing that swimming speed increased with temperature until ∼40°C, then plateaued. These findings are consistent with the observed heterogeneity of B. subtilis populations, and represent the highest reported temperature for swimming in this species. This technique is a simple, low-cost method for quantifying motility at high temperatures and could be useful for investigation of many different cell types, including thermophilic archaea.

Microscopic Object Classification through Passive Motion Observations with Holographic Microscopy.

Digital holographic microscopy provides the ability to observe throughout a volume that is large compared to its resolution without the need to actively refocus to capture the entire volume. This enables simultaneous observations of large numbers of small objects within such a volume. We have constructed a microscope that can observe a volume of 0.4 µm × 0.4 µm × 1.0 µm with submicrometer resolution (in xy) and 2 µm resolution (in z) for observation of microorganisms and minerals in liquid environments on Earth and on potential planetary missions. Because environmental samples are likely to contain mixtures of inorganics and microorganisms of comparable sizes near the resolution limit of the instrument, discrimination between living and non-living objects may be difficult. The active motion of motile organisms can be used to readily distinguish them from non-motile objects (live or inorganic), but additional methods are required to distinguish non-motile organisms and inorganic objects that are of comparable size but different composition and structure. We demonstrate the use of passive motion to make this discrimination by evaluating diffusion and buoyancy characteristics of cells, styrene beads, alumina particles, and gas-filled vesicles of micron scale in the field of view.

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.

The influence of spaceflight and simulated microgravity on bacterial motility and chemotaxis.

As interest in space exploration rises, there is a growing need to quantify the impact of microgravity on the growth, survival, and adaptation of microorganisms, including those responsible for astronaut illness. Motility is a key microbial behavior that plays important roles in nutrient assimilation, tissue localization and invasion, pathogenicity, biofilm formation, and ultimately survival. Very few studies have specifically looked at the effects of microgravity on the phenotypes of microbial motility. However, genomic and transcriptomic studies give a broad general picture of overall gene expression that can be used to predict motility phenotypes based upon selected genes, such as those responsible for flagellar synthesis and function and/or taxis. In this review, we focus on specific strains of Gram-negative bacteria that have been the most studied in this context. We begin with a discussion of Earth-based microgravity simulation systems and how they may affect the genes and phenotypes of interest. We then summarize results from both Earth- and space-based systems showing effects of microgravity on motility-related genes and phenotypes.

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.

Characterization of Retinol Stabilized in Phosphatidylcholine Vesicles with and without Antioxidants.

Retinol stability has been reported to be improved by encapsulation in liposomes, both with and without cholesterol. However, this improvement is limited because of lipid peroxidation. In this study, we compare the stability of retinol in phosphatidylcholine liposomes under ultraviolet (UV) light or standard room air, with and without the addition of antioxidants. Both butylated hydroxytoluene (BHT) and a proprietary mix (StoppOx) improved the shelf stability from <10 to over 30 d. The addition of cholesterol had no effect. Fluorescence imaging showed a heterogeneous distribution of retinol within the vesicles, including within the aqueous layer. Fluorescence lifetimes were equally heterogeneous. Under UV irradiation, StoppOx protected retinol for significantly longer than BHT and via different mechanisms. This suggests that natural antioxidants work well to improve the retinol stability, but that further work to determine the optimal vesicle structure remains to be performed.

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.

Performance of Fluorescent Cell-Labeling Dyes under Simulated Europa Mission Radiation Doses.

We investigated the performance of several commonly used fluorescent dyes after exposure to a simulated Europa mission total ionizing radiation dose of 300 krad (3 kGy) applied using a 60Co source. Dyes irradiated in aqueous solution or as lyophilized powders were evaluated for absorbance and emission spectra, quantum yield, and where appropriate, ability to label cells or nucleic acids. Although some dyes showed significant increase or decrease in quantum yield with the dose, their spectra and cell-labeling properties remained essentially unchanged after irradiation in powder form. Irradiation in aqueous solution led to significantly greater changes, including a large blue shift in the DNA intercalator propidium iodide. These results suggest that many fluorescent probes are appropriate for use in astrobiological missions to Europa, but that SYTO9 and propidium iodide should be used with caution or not mixed with each other, as is commonly done in "Live/Dead" labeling applications.

Scintillation Yield Estimates of Colloidal Cerium-Doped LaF3 Nanoparticles and Potential for "Deep PDT".

A hybrid of radiotherapy and photodynamic therapy (PDT) has been proposed in previously reported studies. This approach utilizes scintillating nanoparticles to transfer energy to attached photosensitizers, thus generating singlet oxygen for local killing of malignant cells. Its effectiveness strongly depends upon the scintillation yield of the nanoparticles. Using a liquid scintillator as a reference standard, we estimated the scintillation yield of Ce0.1La0.9F3/LaF3 core/shell nanoparticles at 28.9 mg/ml in water to be 350 photons/MeV under orthovoltage X-ray irradiation. The subsequent singlet oxygen production for a 60 Gy cumulative dose to cells was estimated to be four orders of magnitude lower than the "Niedre killing dose," used as a target value for effective cell killing. Without significant improvements in the radioluminescence properties of the nanoparticles, this approach to "deep PDT" is likely to be ineffective. Additional considerations and alternatives to singlet oxygen are discussed.

Wavelet-based tracking of bacteria in unreconstructed off-axis holograms.

We propose an automated wavelet-based method of tracking particles in unreconstructed off-axis holograms to provide rough estimates of the presence of motion and particle trajectories in digital holographic microscopy (DHM) time series. The wavelet transform modulus maxima segmentation method is adapted and tailored to extract Airy-like diffraction disks, which represent bacteria, from DHM time series. In this exploratory analysis, the method shows potential for estimating bacterial tracks in low-particle-density time series, based on a preliminary analysis of both living and dead Serratia marcescens, and for rapidly providing a single-bit answer to whether a sample chamber contains living or dead microbes or is empty.

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.

Compact, lensless digital holographic microscope for remote microbiology.

In situ investigation of microbial life in extreme environments can be carried out with microscopes capable of imaging 3-dimensional volumes and tracking particle motion. Here we present a lensless digital holographic microscope approach that provides roughly 1.5 micron resolution in a compact, robust package suitable for remote deployment. High resolution is achieved by generating high numerical-aperture input beams with radial gradient-index rod lenses. The ability to detect and track prokaryotes was explored using bacterial strains of two different sizes. In the larger strain, a variety of motions were seen, while the smaller strain was used to demonstrate a detection capability down to micron scales.

Microbial Morphology and Motility as Biosignatures for Outer Planet Missions.

Meaningful motion is an unambiguous biosignature, but because life in the Solar System is most likely to be microbial, the question is whether such motion may be detected effectively on the micrometer scale. Recent results on microbial motility in various Earth environments have provided insight into the physics and biology that determine whether and how microorganisms as small as bacteria and archaea swim, under which conditions, and at which speeds. These discoveries have not yet been reviewed in an astrobiological context. This paper discusses these findings in the context of Earth analog environments and environments expected to be encountered in the outer Solar System, particularly the jovian and saturnian moons. We also review the imaging technologies capable of recording motility of submicrometer-sized organisms and discuss how an instrument would interface with several types of sample-collection strategies. Key Words: In situ measurement-Biosignatures-Microbiology-Europa-Ice. Astrobiology 16, 755-774.