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

Abstract

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.

Multiwavelength Digital Holographic Imaging and Phase Unwrapping of Protozoa Using Custom Fiji Plug-ins

“…Digital holographic microscopy (DHM) is an emerging technique for 3-dimensional imaging and tracking of microorganisms [1][2][3][4][5] and for in vitro analysis of cellular and subcellular processes such as ion channel function [6] and apoptosis [7,8]. Digital holograms measure the interference of a light wave that has been diffracted through a sample and combined with a coherent, non-diffracted reference wave.…”

“…Dual-or triple-wavelength DHM has been described numerous times as a method for reducing 2π ambiguities in phase imaging [15][16][17]. If a hologram is recorded at two wavelengths, λ 1 and λ 2 , then two independent phase maps are obtained according to Equation (4). If the phase images are subtracted, the resulting image is the equivalent of recording the phase using a "synthetic" or "beat" wavelength :…”

Estimating Carbon Flux From Optically Recording Total Particle Volume at Depths Below the Primary Pycnocline

“…More recently, recordings have been made digitally, and some systems are available commercially (Stemmann et al, 2004 and references listed in their Table 1; Cowen and Guigand, 2008;Ohman et al, 2019). Other imaging systems rely on holography in various configurations (inline and off-axis), with their own advantages (e.g., large depth of field and survey volumes) and disadvantages (e.g., additional processing for image reconstruction) (Malkiel et al, 1999;Watson et al, 2001;Xu et al, 2001;Bochdansky et al, 2013;Lindensmith et al, 2016).…”

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

“…This device eliminates noise due to mechanical and light instabilities, which is necessary for field imaging in harsh conditions [3,5]. However, speckle noise is inherent to the source coherence required to create high-contrast fringes across the array, and stabilization issues in imaging can introduce inconsistencies in amplitude across the image.…”