Digital Holographic Microscopy, a Method for Detection of Microorganisms in Plume Samples from Enceladus and Other Icy Worlds.
ABSTRACT: 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.
Project description: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.
Project description:Self-interference digital holographic microscopy (DHM) has been found particular suitable for simplified quantitative phase imaging of living cells. However, a main drawback of the self-interference DHM principle are scattering patterns that are induced by the coherent nature of the laser light which affect the resolution for detection of optical path length changes. We present a simple and efficient technique for the reduction of coherent disturbances in quantitative phase images. Therefore, amplitude and phase of the sample illumination are modulated by an electrically focus tunable lens. The proposed method is in particular convenient with the self-interference DHM concept. Results from the characterization of the method show that a reduction of coherence induced disturbances up to 70 percent can be achieved. Finally, the performance for enhanced quantitative imaging of living cells is demonstrated.
Project description:BACKGROUND: Digital holography provides a non-invasive measurement of the quantitative phase shifts induced by cells in culture, which can be related to cell volume changes. It has been shown previously that regulation of cell volume, in particular as it relates to ionic homeostasis, is crucially involved in the activation/inactivation of the cell death processes. We thus present here an application of digital holographic microscopy (DHM) dedicated to early and label-free detection of cell death. METHODS AND FINDINGS: We provide quantitative measurements of phase signal obtained on mouse cortical neurons, and caused by early neuronal cell volume regulation triggered by excitotoxic concentrations of L-glutamate. We show that the efficiency of this early regulation of cell volume detected by DHM, is correlated with the occurrence of subsequent neuronal death assessed with the widely accepted trypan blue method for detection of cell viability. CONCLUSIONS: The determination of the phase signal by DHM provides a simple and rapid optical method for the early detection of cell death.
Project description:Ricin and abrin are ribosome-inactivating proteins leading to inhibition of protein synthesis and cell death. These toxins are considered some of the most potent and lethal toxins against which there is no available antidote. Digital holographic microscopy (DHM) is a time-lapse, label-free, and noninvasive imaging technique that can provide phase information on morphological features of cells. In this study, we employed DHM to evaluate the morphological changes of cell lines during ricin and abrin intoxication. We showed that the effect of these toxins is characterized by a decrease in cell confluence and changes in morphological parameters such as cell area, perimeter, irregularity, and roughness. In addition, changes in optical parameters such as phase-shift, optical thickness, and effective-calculated volume were observed. These effects were completely inhibited by specific neutralizing antibodies. An enhanced intoxication effect was observed for preadherent compared to adherent cells, as was detected in early morphology changes and confirmed by annexin V/propidium iodide (PI) apoptosis assay. Detection of the dynamic changes in cell morphology at initial stages of cell intoxication by DHM emphasizes the highly sensitive and rapid nature of this method, allowing the early detection of active toxins.
Project description:Digital holographic microscopy (DHM) has its intrinsic ability to refocusing a sample by numerically propagating an object wave from its hologram plane to its image plane. In this paper opposite-view digital holographic microscopy (OV-DHM) is demonstrated for autofocusing, namely, digitally determining the location of the image plane, and refocusing the object wave without human intervention. In OV-DHM, a specimen is illuminated from two sides in a 4?-alike configuration, and two holograms are generated and recorded by a CCD camera along two orthogonal polarization orientations. The image plane of the sample is determined by finding the minimal variation between the two object waves, and consequently refocusing is performed by propagating the waves to the image plane. Furthermore, the field of view (FOV) of OV-DHM can be extended by combining the two object waves which have an angle in-between. The proposed technique also has the potential to reduce speckle noise and out-of-focus background.
Project description:3D cell culture assays are becoming increasingly popular due to their higher resemblance to tissue environment. These provide an increased complexity compared to the growth on 2D surface and therefore allow studies of advanced cellular properties such as invasion. We report here on the use of 3D Matrigel cell preparations combined with a particular gentle and informative type of live-cell microscopy: quantitative digital holographic microscopy (DHM), here performed by a commercial software-integrated system, currently mostly used for 2D cell culture preparations. By demonstrating this compatibility, we highlight the possible time-efficient quantitative analysis obtained by using a commercial software-integrated DHM system, also for cells in a more advanced 3D culture environment. Further, we demonstrate two very different examples making use of this advantage by performing quantitative DHM analysis of: (1) wound closure cell monolayer Matrigel invasion assay and (2) Matrigel-trapped single and clumps of suspension cells. For both these, we benefited from the autofocus functionality of digital phase holographic imaging to obtain 3D information for cells migrating in a 3D environment. For the latter, we demonstrate that it is possible to quantitatively measure tumourigenic properties like growth of cell clump (or spheroid) over time, as well as single-cell invasion out of cell clump and into the surrounding extracellular matrix. Overall, our findings highlight several possibilities for 3D digital holographic microscopy applications combined with 3D cell preparations, therein studies of drug response or genetic alterations on invasion capacity as well as on tumour growth and metastasis.
Project description:Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive index and to cell thickness. DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes. In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays.
Project description:With the discovery of the persistent jets of water being ejected to space from Enceladus, an understanding of the effect of the space environment on potential organisms and biosignatures in them is necessary for planning life detection missions. We experimentally determine the survivability of microbial cells in liquid medium when ejected into vacuum. Epifluorescence microscopy, using a lipid stain, and SEM imaging were used to interrogate the cellular integrity of E. coli after ejected through a pressurized nozzle into a vacuum chamber. The experimental samples showed a 94% decrease in visible intact E. coli cells but showed a fluorescence residue in the shape of the sublimated droplets that indicated the presence of lipids. The differences in the experimental conditions versus those expected on Enceladus should not change the analog value because the process a sample would undergo when ejected into space was representative. E. coli was selected for testing although other cell types could vary physiologically which would affect their response to a vacuum environment. More testing is needed to determine the dynamic range in concentration of cells expected to survive the plume environment. However, these results suggest that lipids may be directly detectable evidence of life in icy world plumes.
Project description:Enceladus presents an excellent opportunity to detect organic molecules that are relevant for habitability as well as bioorganic molecules that provide evidence for extraterrestrial life because Enceladus' plume is composed of material from the subsurface ocean that has a high habitability potential and significant organic content. A primary challenge is to send instruments to Enceladus that can efficiently sample organic molecules in the plume and analyze for the most relevant molecules with the necessary detection limits. To this end, we present the scientific feasibility and engineering design of the Enceladus Organic Analyzer (EOA) that uses a microfluidic capillary electrophoresis system to provide sensitive detection of a wide range of relevant organic molecules, including amines, amino acids, and carboxylic acids, with ppm plume-detection limits (100 pM limits of detection). Importantly, the design of a capture plate that effectively gathers plume ice particles at encounter velocities from 200?m/s to 5 km/s is described, and the ice particle impact is modeled to demonstrate that material will be efficiently captured without organic decomposition. While the EOA can also operate on a landed mission, the relative technical ease of a fly-by mission to Enceladus, the possibility to nondestructively capture pristine samples from deep within the Enceladus ocean, plus the high sensitivity of the EOA instrument for molecules of bioorganic relevance for life detection argue for the inclusion of EOA on Enceladus missions. Key Words: Lab-on-a-chip-Organic biomarkers-Life detection-Planetary exploration. Astrobiology 17, 902-912.
Project description:We describe an optimized digital holographic microscopy system (DHM) suitable for high-resolution visualization of living cells under conditions of altered macroscopic mechanical forces such as those that arise from changes in gravitational force. Experiments were performed on both a ground-based microgravity simulation platform known as the random positioning machine (RPM) as well as during a parabolic flight campaign (PFC). Under these conditions the DHM system proved to be robust and reliable. In addition, the stability of the system during disturbances in gravitational force was further enhanced by implementing post-processing algorithms that best exploit the intrinsic advantages of DHM for hologram autofocusing and subsequent image registration. Preliminary results obtained in the form of series of phase images point towards sensible changes of cytoarchitecture under states of altered gravity.