Table 1.
Extreme environmental characteristics of the sites sampled.
Fig 1.
Locations include Newport Beach, CA; Malene Bay, Greenland; Utqiaġvik, AK; Salton Sea, CA; Badwater Basin Death Valley, CA; Ash Meadows, NV; Mt. St. Helens, WA; and The Cedars, CA. “North America laea relief location map” by Uwe Dedering is licensed under CC BY-SA 3.0. https://commons.wikimedia.org/wiki/File:North_America_laea_relief_location_map.jpg.
Fig 2.
Instrument deployments at field sites.
(A) Deployment in a sackhole of sea ice brine (Nuuk, Greenland). (B) Deployment in a permafrost tunnel providing borehole access to cryopegs (Utqiaġvik, AK). (C) Deployment in the Mt. St. Helens crater and (D) the Mt. St. Helens glacier caves with recordings performed at ambient air temperature.
Fig 3.
General data processing protocols used to characterize samples. A) Raw hologram with fringe pattern shown in magnified inset. B) The real or imaginary signal is selected in the Fourier plane, shown in the red circle. C) The angular spectrum method is used to reconstruct the data into multiple amplitude or phase z-planes. D) Several different image processing algorithms may be used on the reconstructed images; in most cases, median subtraction is sufficient. E) Z-projection may be used in sparse samples to obtain all of the 3D information in a 2D time series. F) A combination of automated and manual tracking is used to obtain tracks of motile cells.
Fig 4.
(A) Fast swimming, 100 µm/s, is clearly differentiated from Brownian motion even at short time scales of recording. (B) Moderate swimming speeds of 10 µm/s are still many times faster than Brownian motion of single cells and can be differentiated from Brownian motion of aggregates by displacement vs. time curves. (C) The slower swimming and diffusion rates seen at low temperatures can still be readily distinguished, although longer recordings are helpful to capture the shape of the curves.
Fig 5.
Biosignatures from sea ice brine and seawater.
(A) Amplitude image of non-motile diatom from Greenland sea ice brine. Note the demarcation of cell walls and organelles. (B, C) Rapidly swimming organism in amplitude (B) and phase (C) from Greenland sea ice brine (images A-C previously published in [27]). (D) Motile tracks of small microbes from Greenland seawater incubated overnight at 4ºC. Total duration = 300 frames or 20 s. Tracks were obtained from a sum of amplitude reconstructions taken every 5 μm over 500 μm depth and tracked using TrackMate automated detection and LAP tracking.
Fig 6.
Biosignatures in the Salton Sea hot spring.
(A-C) Amplitude reconstructions of large microbes. (A) Large microbe with dark absorption characteristic of chlorophyll. (B) Elongated filaments without chlorophyll. (C) Arrangement of several filaments with a cloud of putative cells/organics. (D-F) HELM motion history images (MHI) with color-coding indicating time lapse of selected areas at different temperatures. (D) At 25ºC, a single motile small microbe is observed making a complex trajectory in the imaging volume over a 675 frame (45 s) recording period. (E) At least 3 highly motile small microbes are seen in 35 s at 30ºC. (F) At 42ºC, the large microbes are highly active, though moving slowly as this 45 s recording period shows (the upper part of this panel corresponds to Panel C). (G, H) Chemotaxis and possible predation. (G) A needle filled with serine at the beginning of the recording. (H) Panel G after 60 min. Blue is a maximum projection of the frame-to-frame changes over the recording period. The arrow indicates a motile large microbe possibly preying on the small microbial cloud.
Fig 7.
Biosignatures from Death Valley/Ash Meadows.
(A) Amplitude (left) and phase (right) images of a motile (gliding) diatom from Badwater Basin Pool. (B) Amplitude (left) and phase (right) images of a motile ciliate from Ash Meadows. (C) MHI of an in situ recording showing the run-reverse swimming pattern of two organisms from Badwater basin in Death Valley. (D) Manually tracked trajectories of the motile organisms in (C).
Fig 8.
Biosignatures from glacier meltwater from Crater Glacier, Mt. St. Helens.
(A) Spindle-shaped microminerals, amplitude reconstruction on the focal plane of the bottom of the sample chamber. (B) Amplitude reconstruction 80 μm above the bottom of the chamber, showing a motile organism (arrow) and some suspended non-motile particles with significantly higher contrast (S14 Video). (C) Phase image 80 μm above the bottom of the chamber, showing a motile organism (arrow) with low phase contrast surrounded by microminerals of high contrast.
Fig 9.
Biosignatures from the Mt. St.
Helens crater. (A-C) Orange biofilm sludge. (A) Orange biofilm sludge amplitude reconstruction; the circle indicates a motile cell. (B) Orange biofilm sludge phase reconstruction, showing the phase shift contrast between the motile cell (circled) and microminerals (arrow). (C) 2D tracks of frame-to-frame subtracted reconstruction of glacier discharge showing several motile small microbes. (D-G) Midstream. (D) Context photo of photosynthetic cyanobacterial benthic mats. (E) Benthic mat DHM amplitude reconstruction at z = 0 (approximately halfway through the 1 mm deep sample chamber). (F, G) MHI at two different depths from the benthic mat, showing rapidly swimming organisms at very different focal planes.
Fig 10.
Helens glacial cave ice samples. (A, B) MHI images showing motion of a single recording conducted in situ immediately after ice melted. All motile small microbes in glacial ice samples had low SNR and were therefore difficult to identify in MHI. Arrows in each image indicate an observable trajectory. Both MHIs were created using reconstructed amplitude images where each image in the time series was the z-plane where the microbe of interest was in focus. This approach allowed the MHI to detect these two trajectories. (C) MATLAB plot showing the corkscrew trajectory of the motile small microbe shown in the bottom left of (A), (D). (D) MATLAB plot showing manually tracked 2D trajectories of all six motile small microbes observed in this recording.
Fig 11.
Motility and morphology from The Cedars site.
(A) Selected trajectory of a motile microorganism found in the direct GPS1 spring sample. This image is a composite image rendered by the minimum pixel intensity projection of 7 s of data. (B) A single plane intensity reconstruction of a select micrometer-sized organism from the BS5 spring sample. (C) MATLAB 2D plot of two tracked motile small microbes from the BS5 pool.
Fig 12.
Morphology and motility biosignatures from cryopeg brine samples.
(A) A single plane intensity reconstruction of a select micrometer-sized organism in a brine sample at 17.5°C. The arrows in (A) and (B) are identifying the same motile low contrast small microbe from the same recording. (B) A MATLAB 2D plot of small microbial motile organisms, showing trajectories of motile organisms at 17.5°C in a cryopeg brine sample from a thermal gradient experiment.
Table 2.
Summary of data analysis: sample types and motility observed.