Manuel Bedrossian

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.

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 instruments 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 DHMs 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 DHMs 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.

Sources and propagation of errors in quantitative phase imaging techniques using optical interferometry

Quantitative phase imaging (QPI) has many applications in a broad range of disciplines from astronomy to microbiology. QPI is often performed by optical interferometry, where two coherent beams of light are used to produce interference patterns at a detector plane. Many algorithms exist to calculate the phase of the incident light from these recorded interference patterns as well as enhance their quality by various de-noising methods. Many of these de-noising algorithms, however, corrupt the quantitative aspect of the measurement, resulting in phase contrast images. Among these phase calculation techniques and de-noising algorithms, none approach the optimization of phase measurements by theoretically addressing the various sources of error in its measurement, as well as how these errors propagate to the phase calculations. In this work, we investigate the various sources of error in the measurements required for QPI, as well as theoretically derive the influence of each source of error on the overall phase calculation for three common phase calculation techniques: the four bucket/step method, three bucket/step method, and the Carré method. The noise characteristics of each of these techniques are discussed and compared using error parameters of a readily available CCD sensor array. Additionally, experimental analysis is conducted on interferograms to investigate the influence of speckle noise on the phase measurements of the three algorithms discussed.

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.

Imaging technologies and strategies for detection of extant extraterrestrial microorganisms

Abstract There is no reductionist definition of life, so the way organisms look, behave, and move is the most definitive way to identify extraterrestrial life. Life elsewhere in the Solar System is likely to be microbial, but no microscope capable of imaging prokaryotic life has ever flown on a lander mission to a habitable planet. Nonetheless, high-resolution microscopes have been developed that are appropriate for planetary exploration. Traditional light microscopy, interferometric microscopy, light-field microscopy, scanning probe microscopy, and electron microscopy are all possible techniques for the detection of extant micro-organisms on Mars and the moons of Jupiter and Saturn. This article begins with a general discussion of the challenges involved in searching for prokaryotic life, then reviews instruments that have flown, that have been selected for flight but not flown or not flown yet, and developing techniques of great promise for life detection that have not yet been selected for flight.

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.