Data from: Extant life detection using label-free video microscopy in analog aquatic environments

Snyder, Carl 1 ; Nadeau, Jay 1 ; Bedrossian, Manuel2; Barr, Casey3; Deming, Jody4; Lindensmith, Christian5; Stenner, Christian6

Published Mar 17, 2026 on Dryad. https://doi.org/10.5061/dryad.70rxwdc41

Abstract

The ability of microbial morphology, active motion, and refractive index to serve as biosignatures was investigated by in situ video microscopy in a wide range of extreme field sites where such imaging had not previously been performed. These sites allowed for sampling seawater, sea ice brines, cryopeg brines, hypersaline pools and seeps, hyperalkaline springs, and glaciovolcanic cave ice. In all samples, except the cryopeg brine, active motion was observed without any sample treatment. Active motion was observed in the cryopeg brines when samples were subjected to temperature gradient above in situ. Levels of prokaryotic motility were, in general, low in the field samples collected at temperatures < 4ºC. Non-motile cells could be distinguished from microminerals by differences in passive motion (e.g., density measured by sinking/floating), refractive index and/or absorbance, or morphology in the case of larger eukaryotes. Dramatic increases in the fraction of motile cells were seen with simple stimuli such as warming or the addition of L-serine. Chemotaxis and thermotaxis were also observed in select samples. An open-source, autonomous software package with computational requirements that can be scaled to spaceflight computers was used to classify the data. These results demonstrate the utility of volumetric light microscopy for life detection, but also suggest the importance of developing methods to stimulate cells in situ and process data using the restrictions imposed by mission bandwidth, as well as instruments to capture cell-like objects for detailed chemical analysis.

https://doi.org/10.5061/dryad.70rxwdc41

Overview

This data package contains raw digital holographic microscopy (DHM) video recordings collected from a range of extreme aquatic field environments across western North America. These recordings were used to investigate whether active microbial motion, cell morphology, and optical properties can serve as biosignatures of extant life detectable by label-free video microscopy. Data are provided in support of the associated publication in PLoS ONE (DOI: 10.1371/journal.pone.0318239).

All videos are raw hologram recordings acquired by a custom off-axis DHM instrument (SHAMU or Son of SHAMU) operating at a 405 nm laser wavelength, with a maximum acquisition rate of 15 frames per second. Each hologram video can be reconstructed into amplitude and phase image stacks using the angular spectrum method via the associated Fiji plug-ins described in the publication.

File Naming Convention

Most field sample video files follow this general naming pattern:

[Location]_[SampleType]_[YYYY.MM.DD]_[HH-MM]_Holograms.avi

Each underscore-delimited segment encodes a specific piece of metadata about the recording:

[Location]: A text abbreviation identifying the geographic location of sample collection (see Section 3 for all codes).
[SampleType]: A text descriptor identifying the type of aquatic sample collected (see Section 3 for all codes).
[YYYY.MM.DD]: The date of recording in Year.Month.Day format — for example, 2017.04.21 indicates 21 April 2017.
[HH-MM]: The time of recording in 24-hour format, with a hyphen separating hours and minutes — for example, 11-12 indicates 11:12.
Holograms: A literal text marker indicating that the file contains raw hologram frames that require computational reconstruction before the microscopy data can be interpreted.
.avi: Audio Video Interleave container format; video frames are encoded as raw hologram images.

Two files deviate from this pattern. MSH_GreenMatScroll.avi and MSH_GlacierWaterRunOff_2020.09.30_Holograms.avi omit the time segment because only a single recording was taken at each location, making time disambiguation unnecessary. The five Salton Sea thermotaxis experiment files (SS1–SS5) use a different naming convention altogether, described in Section 4.

Location and Sample Type Codes

The following codes appear in the first two segments of each field recording filename. Each entry gives the location abbreviation, the full location name, and the sample type codes used for that site.

AM — Ash Meadows National Wildlife Refuge, Nevada

AM_KingsPool: Hypersaline spring-fed pool (Kings Pool); ambient temperature ~20°C.

Barrow — Utqiġvik (Barrow), Alaska

Barrow_Cryopeg: Ancient permafrost-encased brine layer (cryopeg); sampled at −6°C in situ.
Barrow_Sea_Water: Seawater collected near Utqiġvik.
Barrow_SeaIceBrine: Brine extracted from sea ice sackhole channels.

DeathValley — Badwater Basin, Death Valley, California

DeathValley_BWSpring: Hypersaline pool/spring at Badwater Basin.

MSH — Mt. St. Helens Crater, Washington

MSH_DownStream: Water downstream from the hot spring–glacier mixing zone; predominantly large eukaryotes.
MSH_GlacierWaterRunOff: Discharge from Crater Glacier surface melt (~0.7°C); high mineral content.
MSH_GreenMatScroll: Sample from the dense photosynthetic cyanobacterial benthic mat at the midstream site.
MSH_MidStream: Midstream zone where hot spring water mixes with glacier discharge (30–37°C).

Salton Sea Thermotaxis Experiment Files (SS1–SS5)

Five files from the Salton Sea hot spring site use a distinct naming convention. Rather than encoding location and date, these filenames reflect a controlled temperature-ramp motility experiment: “SS” stands for Salton Sea, the number indicates the sequential experimental step, and “_[T]C” denotes the set temperature in degrees Celsius. SS5 substitutes a descriptive behavioral label for the temperature because it captures a dynamic phenomenon rather than a single temperature point.

SS1_22C.avi — Room temperature baseline (22°C). No motility was apparent in situ.
SS2_25C.avi — Sample warmed to 25°C. Sparse small microbial motility observed (~1 motile cell per imaging volume).
SS3_30C.avi — Sample warmed to 30°C. Markedly increased motility (~40% of particles motile, 4–5 per imaging volume).
SS4_42C.avi — Sample warmed to 42°C. Majority of both small and large microbes motile; large microbes display gliding and grazing behavior.
SS5_TaxisGrazing.avi — Time-lapse chemotaxis recording (~30–42°C). An L-serine-loaded agarose needle was inserted into the sample; the recording documents progressive accumulation of small microbes around the needle tip and apparent predation of the microbial cloud by a large microbe.

Complete File List with Descriptions

Ash Meadows, Nevada — Kings Pool

Three recordings were made at Kings Pool on 21 April 2017, at 11:12, 12:26, and 14:25 respectively. All three are hypersaline spring-fed pool samples.

AM_KingsPool_2017.04.21_11-12_Holograms.avi — Kings Pool hypersaline spring, recorded 11:12 on 21 April 2017.
AM_KingsPool_2017.04.21_12-26_Holograms.avi — Kings Pool hypersaline spring, recorded 12:26 on 21 April 2017.
AM_KingsPool_2017.04.21_14-25_Holograms.avi — Kings Pool hypersaline spring, recorded 14:25 on 21 April 2017.

Utqiġvik (Barrow), Alaska

Five recordings were made across three sample types at Utqiġvik: three from the cryopeg brine, one from open seawater, and one from sea ice brine.

Barrow_Cryopeg_2017.05.09_03-10_Holograms.avi — Cryopeg brine from permafrost, recorded 03:10 on 9 May 2017.
Barrow_Cryopeg_2017.05.09_03-12_Holograms.avi — Cryopeg brine from permafrost, recorded 03:12 on 9 May 2017.
Barrow_Cryopeg_2017.05.09_03-15_Holograms.avi — Cryopeg brine from permafrost, recorded 03:15 on 9 May 2017.
Barrow_Sea_Water_2017.05.08_17-37_Holograms.avi — Seawater sample collected near Utqiġvik, recorded 17:37 on 8 May 2017.
Barrow_SeaIceBrine_2017.05.07_19-11_Holograms.avi — Sea ice brine extracted from sackhole channels, recorded 19:11 on 7 May 2017.

Death Valley, California — Badwater Basin

Four recordings were made at Badwater Basin across two days in April 2017.

DeathValley_BWSpring_2017.04.10_15-54_Holograms.avi — Badwater Basin hypersaline pool/spring, recorded 15:54 on 10 April 2017.
DeathValley_BWSpring_2017.04.10_15-59_Holograms.avi — Badwater Basin hypersaline pool/spring, recorded 15:59 on 10 April 2017.
DeathValley_BWSpring_2017.04.11_15-27_Holograms.avi — Badwater Basin hypersaline pool/spring, recorded 15:27 on 11 April 2017.
DeathValley_BWSpring_2017.04.11_15-35_Holograms.avi — Badwater Basin hypersaline pool/spring, recorded 15:35 on 11 April 2017.

Mt. St. Helens Crater, Washington

Eight recordings were made at three distinct sites within the Mt. St. Helens crater during September–October 2020: the downstream zone, the glacier water runoff, the midstream benthic mat, and the midstream mixing zone.

MSH_DownStream_2020.09.30_14-30_Holograms.avi — Downstream site (hot spring + glacier mixing zone), recorded 14:30 on 30 September 2020.
MSH_DownStream_2020.09.30_14-33_Holograms.avi — Downstream site, recorded 14:33 on 30 September 2020.
MSH_DownStream_2020.09.30_14-39_Holograms.avi — Downstream site, recorded 14:39 on 30 September 2020.
MSH_GlacierWaterRunOff_2020.09.30_Holograms.avi — Crater Glacier meltwater discharge (~0.7°C), collected 30 September 2020. A single recording was taken; no time segment is included in the filename.
MSH_GreenMatScroll.avi — Dense photosynthetic cyanobacterial benthic mat at the midstream site. This is a single representative scroll recording through the mat sample; no date or time segment is included in the filename.
MSH_MidStream_2020.10.06_10-58_Holograms.avi — Midstream hot spring–glacier mixing zone, recorded 10:58 on 6 October 2020.
MSH_MidStream_2020.10.13_19-39_Holograms.avi — Midstream site, recorded 19:39 on 13 October 2020.
MSH_MidStream_2020.10.13_19-47_Holograms.avi — Midstream site, recorded 19:47 on 13 October 2020.

Salton Sea, California — Thermotaxis Experiment

Five recordings document a controlled temperature-ramp and chemotaxis experiment. See Section 4 for full descriptions.

SS1_22C.avi — Salton Sea hot spring sample at 22°C (room temperature baseline; no motility).
SS2_25C.avi — Salton Sea sample warmed to 25°C (sparse small microbial motility).
SS3_30C.avi — Salton Sea sample warmed to 30°C (marked increase in motility).
SS4_42C.avi — Salton Sea sample warmed to 42°C (majority of small and large microbes motile; gliding and grazing behavior).
SS5_TaxisGrazing.avi — Salton Sea chemotaxis and grazing time-lapse; L-serine agarose needle inserted into sample.

Access Information

Video player. We use ImageJ or FIJI regularly.

Field Sites and Samples

Sites analyzed ranged across western North America and also included West Greenland (Fig 1). Field sites were visited that exhibited one or more environmental parameters that could be considered extreme: temperature, salinity, pH, oxygen concentration, or light availability. Several of them are known to be useful analogs for potential near-term lander missions, and others are emerging sites of interest. A summary of the sites is given in Table 1, and full descriptions with context images and references to geochemical analyses where available are provided in Supplementary Information text and Fig S1-S5.

Materials and Methods

Digital holographic microscope (DHM). The off-axis DHM used throughout this work has been described previously. Its design has been optimized for easy adaptability to spaceflight (e.g., no compound objective lenses) and for balance between resolution and depth of field in order to obtain a limit of detection of ~10³ bacteria per milliliter. Briefly, a coherent and monochromatic light source (405 nm diode laser) is collimated. This light source is then passed through two microfluidic wells. One well contains a sample, while the other contains a reference liquid in order to match optical path lengths with the sample. The sample and reference beams are then passed through two separate identical aspheric objective lenses (NA = 0.3) and recombined at an image sensor via a relay lens. This instrument is capable of diffraction-limited sub-micrometer resolution. For field use, the optical train is housed in a rugged water-resistant container along with all necessary electronics for the instrument’s stand-alone operation. These include a processor, hard drive, power source, laser, and camera, as well as diagnostic sensors. The acquisition speed is a maximum of 15 frames per second. The first iteration of the instrument, dubbed SHAMU, was first described in and is pictured as deployed in Fig 2A, 2B. A smaller version was developed in 2019, referred to as Son of SHAMU, based upon a smaller pixel pitch camera (2.2 mm vs 3.45 mm), allowing correspondingly reduced relay optics that are folded to create a more compact system (pictures of deployments in Fig 2C, 2D). A comparison of the two instruments is shown in Supporting Information Fig S6. The mass of the Son of SHAMU is 6 kg, reduced from 11 kg. Data are acquired using a custom open-source platform, DHMx. Optical resolution of the two systems is the same.

Sampling and test conditions. For liquid samples from seawater or pools, a sterile container was used to collect liquid, which was injected into the sample chamber using a sterile syringe and imaged immediately at ambient temperature. For recording from sea ice brines, the instrument was placed in a sackhole as in Fig 2A. Brine collected as it seeped from the channels over a time course of 1-2 h, when it was collected with a sterile syringe and injected into the sample chamber. Liquid samples imaged in situ were sometimes filter-concentrated using a syringe with an attached 0.22 µm filter. Ice samples were returned to camp and melted at ambient temperatures (4°C to 8°C) before recordings were taken. Samples that were returned to field laboratories underwent a variety of test conditions according to their provenance.

Sea ice and brine samples from Arctic environments were collected into sterile 50-cc test tubes and incubated in cold rooms at 4°C and –6°C to evaluate thermal response. The amino acid serine, the sugar trehalose or glucose, or a complete rich medium (Difco marine broth 2216) was added to selected samples and incubated from 60 seconds to overnight before imaging.

Samples from The Cedars and Mt. St. Helens were imaged on site at ambient air temperatures (~20ºC and ~13ºC, respectively). Returned samples from environments at or above room temperature were imaged at room temperature and then ramped to higher temperatures using a digitally temperature-controlled microscope stage (Warner Instruments CL-100 temperature controller).

Estimate of cell count by DHM. Each volume of view in the DHM is 0.24 mL, so cell counts are estimated by averaging the number of cells per volume of view over the number of captured frames. In the case of eukaryotic cells, cell counts were often < 1 cell average per frame. Note that 1 cell per frame equals approximately 4000 cells/mL.

Chemotaxis. Chemotaxis was evaluated by creating a reservoir of the stimulant to be measured within a 1% agarose gel. Low-melting point (LMP) agarose (Sigma-Aldrich) was added to 0.9% saline and microwaved until dissolved. After cooling to ~60°C, chemoattractant was added to the desired concentration and the mixture was loaded into a sterile pulled borosilicate pipette tip or 27-gauge needle. This glass pipette was pulled on a Sutter Instruments P-97 puller using “patch clamp” settings. The tip diameter range was 1-5 mm. For experiments conducted in the field where small diameter glass pipettes were too fragile, sterile 27-gauge syringe needles were used to provide the chemoattractant substrate. After the agarose hardened, the substrate was inserted into the sample chamber for imaging.