Concept and development of a multimodal bioanalytic laboratory for the in-situ exploration of icy ocean worlds

The definition of life and its universal attributes remains debated, making extraterrestrial life detection inherently uncertain. Robust analytical strategies therefore should not rely on single techniques, even if highly specific, but instead integrate complementary approaches capable of probing multiple classes of biosignatures. Here we present the concept and prototype development of an automated multimodal in situ bioanalytical laboratory TRIPLE (Technologies for Rapid Ice Penetration and subglacial Lake Exploration) AstroBioLab (ABL), designed as a technology test bed for astrobiological exploration of icy ocean worlds. The system is conceived as a modular scientific payload for the analysis of aqueous samples over a wide volume range (milliliters to liters) and integrates several nodes in the analytical pipeline. These include (i) microscopy enabling bright-field, dark-field, and autofluorescence imaging; (ii) fluorescence spectroscopy; (iii) compact mass spectrometry; and (iv) an optional module for metagenomic analysis. Supporting subsystems enable automated sample handling, decontamination, data processing, storage, and transmission. Together, these capabilities allow structurally, chemically, and molecularly relevant biosignatures to be examined within a unified analytical framework, increasing detection confidence in environments where indicative biosignatures remain uncertain. The study discusses both technological opportunities and foreseeable engineering and operational constraints, including planetary protection considerations and contamination control. The proposed multimodal laboratory architecture represents a scalable strategy for biosignature detection and habitability assessment in future missions to icy ocean worlds.

Icy ocean worlds have emerged as some of the most compelling targets for near-term astrobiological exploration. At the same time, intensive debate continues over how life should be defined and which attributes are universal, making targeted life detection a probabilistic inference task rather than a binary yes/no measurement. Contemporary strategic assessments and agency roadmaps accordingly emphasize astrobiology and ocean worlds as priority themes, and simultaneously stress the need for more sophisticated frameworks that account for potential false positives and false negatives (type I and type II errors) and that explicitly integrate environmental context and multiple independent lines of evidence (Hendrix et al., 2019; Smith and Mathis, 2023).

Among ocean worlds, Enceladus is distinctive because plume activity provides direct access to material sourced from its subsurface ocean without requiring deep subsurface penetration (Waite et al., 2017). Analyses of ejected ice grains and associated components indicate a chemically diverse organic inventory, including macromolecular organic compounds; more recent work reports additional organic compounds in freshly ejected ice grains (Postberg et al., 2018). In parallel, detection of molecular hydrogen in the plume has been interpreted as evidence for ongoing water–rock interactions and associated redox gradients that could sustain metabolisms in hydrothermal settings, highlighting why habitability and biosignature interpretation must be treated as coupled questions rather than separate steps (Waite et al., 2017; Glein et al., 2018).

Europa presents a complementary and, in some respects, more ambiguous context. Surface–ice–ocean coupling and radiation-driven alteration can modify and potentially degrade candidate biosignatures, and synthesis reviews for upcoming Europa exploration emphasize that key uncertainties remain in the composition of surface materials and any transient atmospheric or plume-related constituents (Pappalardo et al., 2024). These uncertainties directly affect which molecular and structural targets are realistic and how confidently ocean-derived signals can be interpreted (Becker et al., 2024). Moreover, emerging geophysical analyses suggest that present-day seafloor activity may be limited, with implications for the persistence and magnitude of chemical disequilibria that might support chemosynthetic ecosystems and, by extension, for the expected strength and variability of biosignature sources (Byrne et al., 2026).

This scientific motivation is increasingly being translated into mission architecture and payload priorities. NASA’s Europa Clipper mission will employ high-sensitivity gas analysis (including advanced mass spectrometry) to characterize gases near Europa and to constrain the chemistry of Europa’s surface, atmosphere, and suspected ocean in support of habitability investigations (Becker et al., 2024; Pappalardo et al., 2024). Complementarily, European Space Agency’s JUICE mission will make detailed observations of Jupiter and its large ocean-bearing moons with a suite of remote sensing, geophysical, and in situ instruments, explicitly framing these bodies as potential habitats and providing system-level context for ocean-world comparisons (Grasset et al., 2013; Fletcher et al., 2023). Flagship-level mission concept studies for Enceladus explicitly motivate payload complementarity and redundancy for life detection and emphasize how architecture and sampling strategy govern the interpretability of both positive and null results (Neveu et al., 2020; MacKenzie et al., 2021).

Despite the field’s enthusiasm for “biosignatures,” the term is not synonymous with life: it denotes an observation that may suggest a biological cause given an environmental context, yet it must always be evaluated against plausible abiotic alternatives and measurement artifacts (Catling et al., 2018; Smith and Mathis, 2023). Recent conceptual analyses argue for clarity and accuracy in how biosignature claims are formulated and reported, while community efforts propose explicit assessment criteria intended to systematize how different biosignature classes contribute to confidence, interpretability, and decision-making under uncertainty (Green et al., 2021). Formal treatments of biosignature evaluation emphasize that “perfect biosignatures” with zero false-positive and zero false-negative probability are unlikely, and that realistic frameworks must explicitly account for signal-versus-noise tradeoffs in the face of sparse statistics and complex, multi-process detection chains (Catling et al., 2018; Schwieterman et al., 2018).

These epistemic requirements translate directly into instrument strategy. Multimodal in situ approaches are attractive because they enable structurally, chemically, and molecularly relevant information to be acquired from the same samples and interpreted as a coherent evidence chain. Imaging offers access to spatial organization and morphology, including the identification of cell-sized structures, microtextures, and particulate populations, while optical spectroscopy reveals fluorophores, functional groups, and mineral-organic associations that contextualize candidate biosignatures. Mass spectrometry complements these observations by delivering sensitive molecular inventories and pattern information (e.g., homologous series, fragmentation behavior, isotopic constraints), thereby supporting both targeted and future agnostic searches in environments where the most diagnostic biosignature classes remain uncertain (Postberg et al., 2018; Arevalo et al., 2020).

Technological trends make this kind of integration increasingly feasible in compact, automated architectures compatible with mission resource constraints. Reviews of lab-on-a-chip and microfluidic technologies for space research highlight how sample capture, concentration, chemical processing, and detection can be integrated into energy-efficient miniaturized systems (Cinti et al., 2023; Krakos, 2023). These capabilities are especially relevant for high-salinity matrices and dilute targets expected for ocean world liquids or melts. Flight-proven Raman/luminescence investigations on Mars (e.g., SHERLOC) illustrate that co-located Raman and fluorescence/luminescence measurements can jointly interrogate organics and their mineral context, providing a useful technical analogue for extending fluorescence pipelines toward future Raman regimes in compact payloads (Rull et al., 2017; Bhartia et al., 2021). For cellular- and particle-scale life detection, flow cytometry with exogenous fluorescent stains has been proposed and experimentally demonstrated as a promising pathway for icy-moon applications, enabling classification and discrimination of biotic and abiotic material while targeting multiple biomarker classes (Nadeau et al., 2018; Dunker et al., 2022). Finally, recent demonstrations of nanopore sequencing and onboard “sample-to-answer” analytics in space operations motivate an optional metagenomic module as a future extension for terrestrial applications or scenarios where nucleic-acid-based life hypotheses are scientifically justified, while simultaneously underscoring the stringent contamination control that such approaches require (Chen et al., 2020; Jia et al., 2023; Sharma et al., 2025).

This study is embedded in the project line “Technologies for Rapid Ice Penetration and subglacial Lake Exploration” (TRIPLE), supported by the German Space Agency at DLR. This program aims to develop modular technologies for accessing and investigating subglacial aquatic environments as terrestrial analogs for icy ocean worlds. Building on this scientific and technological foundation, we present the concept and prototype development of the automated multimodal in situ bioanalytical laboratory TRIPLE AstroBioLab (TRIPLE ABL), intended as a technology-development test bed for future exploration under icy-moon-relevant conditions.

The TRIPLE ABL design is shaped by five intertwined drivers. First, the science driver: rather than committing to a single biosignature class, the payload is built to investigate structural (cellular morphology and motility), chemical (autofluorescence of CDOM and biologically relevant fluorophores, CHNOPS inventories, and chemical disequilibria), and molecular (nucleic acid) lines of evidence on the same sample (Catling et al., 2018; Neveu et al., 2018). Second, modularity at both hardware and software levels: each analytical pipeline is implemented as an independent subsystem with its own controller and a common interface, so that individual modules can be substituted, upgraded, or removed without re-engineering the whole payload. Third, an explicit resource envelope: the prototype is bounded by a single expedition-grade housing, a powerful Li-ion reservoir with optional 400 W photovoltaic augmentation, and a predominantly 12 V DC architecture, providing a tractable analogue for the mass, power, and volume constraints that a future flight payload might utilize after further miniaturization. Fourth, environmental robustness: an active cascading thermal architecture, thermal insulation, vibration-isolated structural subunits, and a UV-C decontamination cycle are dimensioned to maintain thermally controlled internal environment for sensitive optics and chemistry while the box itself is exposed to subzero ambient temperatures and the mechanical handling typical of polar field operations (Coustenis et al., 2023). Fifth, an automation philosophy that intentionally separates fully autonomous, non-destructive screening from a deliberate human-in-the-loop transition into any destructive measurement. Since subglacial samples are scarce and irreplaceable, and because life-detection claims are particularly sensitive to operator decisions taken upstream of the data, the system prioritizes sample conservation, traceability, and analyst oversight over raw throughput.

System overview and design principles of the TRIPLE ABL

The TRIPLE ABL system (in particular the current version developed for the Antarctic mission at Neumayer Station III) is designed as a modular scientific payload for aqueous sample analysis across a broad volume range and integrates multiple parallel analytical pipelines: (i) microscopy supporting bright-field, dark-field, and (auto)fluorescence imaging; (ii) fluorescence spectroscopy; (iii) a compact mass spectrometry module; and (iv) an optional module for metagenomic analysis (Figure 1).

Microscopy is implemented in TRIPLE ABL through three complementary modalities: bright-field, dark-field, and fluorescence microscopy. Bright-field imaging provides general cell morphology, dark-field enables visualization of membranes and transparent microorganisms, and fluorescence imaging enables the detection of naturally fluorescent biological or biomolecular features, including chlorophyll-containing microorganisms, where present. Together, these modalities provide complementary and partially overlapping information, supporting the characterization of microbial cells, particles, and morphologically relevant structures across a range of biological and physicochemical properties.

Fluorescence spectroscopy is employed in the TRIPLE ABL framework to detect optical signatures potentially associated with biological activity. Biomolecules such as NADH, FAD, ATP, chlorophyll, and tryptophan exhibit characteristic excitation–emission behavior, which can support their identification under suitable measurement conditions. Because no exogenous dyes or stains, such as Texas Red or DAPI, are introduced in this non-destructive analytical block, the detection principle relies on native optical phenomena, including absorption, scattering, refraction, and autofluorescence. This helps preserve the chemical integrity of the sample for subsequent mass spectrometry and optional metagenomic analysis.

Within the “destructive” analytical block, mass spectrometry serves as a high-sensitivity tool for detecting volatile compounds, molecular inventories, and potential metabolic gases in extreme subglacial environments. Deploying this capability in terrestrial field analogs is important for establishing operational heritage and technological foundations for future life-detection missions targeting subsurface oceans of icy moons (Waite et al., 2024). Finally, metagenomic analysis offers an optional means of characterizing terrestrial or terrestrial-like DNA-based life by supporting taxonomic identification and functional or metabolic inference, particularly in combination with microscopy and other analytical modules. In addition to these analytical modalities, the TRIPLE ABL concept includes routines for autonomous data storage, processing, and prioritized transmission, enabling adaptive measurement sequencing under communication latency and bandwidth constraints typical of outer Solar System missions.

Contamination and cross-contamination control are treated as key design drivers. COSPAR planetary protection policy aims to prevent forward contamination of targets of biological interest and to preserve the scientific integrity of life-detection investigations. It specifies mission-category-dependent implementation guidelines, including stringent cleanroom practices, documentation of contamination-control measures, and bioburden reduction or sterilization approaches where appropriate (Coustenis et al., 2023). Accordingly, the TRIPLE ABL design emphasizes contamination-minimizing sample pathways and processing routines as enabling conditions for interpretable multimodal biosignature assessment in environments where life-detection criteria remain uncertain.

Housing and thermal management features of the TRIPLE ABL

The TRIPLE ABL in current configuration is divided into two physically separated units to improve operational handling, weight distribution, and vibration isolation. The main analytical unit (ABL 01.1–0) is housed in a customized aluminum expedition case (Zarges GmbH, Weilheim, Germany) and contains the fluidic system, optical instrumentation (spectroscopy and microscopy), computing core, and power-management electronics (Figure 2). The mass spectrometry unit (ABL 01.2–0) is housed separately in a ruggedized case manufactured by Peli Products GmbH, Crottendorf, Germany). This separated architecture reduces mechanical coupling between the sensitive vacuum and ionization components of the mass spectrometer and the vibrations generated by the fluidic pumps of the main analytical unit.

The thermal management system is designed to stabilize the laboratory compartment at approximately 22 °C. Heat is generated by four positive-temperature-coefficient (PTC) heating elements (12 V, 15 W) mounted within the aluminum structural frame, which also acts as a passive heat-distribution element. Thermal monitoring is performed using a distributed sensor array consisting of three internal and one external (mounted to the outside aluminum box) air-temperature sensors used to characterize thermal gradients within the chamber volume, together with four direct-contact sensors positioned adjacent to the heating elements on the structural frame. Surface temperatures near the heating elements are monitored using negative-temperature-coefficient (NTC) sensors, while MCP9808 sensors are used for compartment air-temperature measurements, together providing spatially distributed thermal feedback for closed-loop control.

One of the major engineering challenges is maintaining a thermally controlled environment for fluidic and analytical processes while operating on the Antarctic ice shelf. The TRIPLE ABL therefore employs an active cascaded PID-MIMO (Proportional-Integral-Derivative, Multiple-Input Multiple-Output) thermal-control strategy. The control logic includes a hard safety limit that prevents heater surface temperatures from exceeding 40 °C. This constraint is implemented to avoid localized thermal hotspots that could alter or damage samples prior to analysis and to prevent structural softening of ABL 01.1–0 components fabricated from polylactic acid (PLA). Additional internal heat sources generated by electronic subsystems are incorporated into the thermal-control model.

For thermal insulation, the inner housing of TRIPLE ABL 01.1–0 is lined with 50 mm expanded polystyrene (EPS) insulation with a thermal conductivity of approximately 0.04 W m -1 K −1 (Gibson and Ashby, 1997). The insulation layer is additionally covered with aluminized sheets (W. Söhngen GmbH) to reduce passive heat loss.

Power management and field autonomy

The TRIPLE ABL system is designed for hybrid operation using both renewable energy sources and station-based power infrastructure. A high-capacity 1,008 Wh lithium-ion battery (Anker Innovations Ltd. Anker C1000 Gen2) serves as the central power reservoir, delivering up to 2000 W continuous output with 3000 W peak power capability. Although the battery system supports 220 V AC output, most internal subsystems, including pumps, illumination units, and sensor electronics, are designed to operate directly from a 12 V DC power bus to minimize conversion losses associated with voltage inversion.

During field deployment, the system can additionally be supported by a portable 400 W solar array. Under typical analytical operation without substantial external heating demand, the TRIPLE ABL is designed to maintain thermal stability and operational functionality for approximately 10 h on a single battery charge. For extended deployment campaigns at Neumayer Station III, the system is intended to operate using the station’s generator-based power grid.

Control and communication design

The internal architecture of the TRIPLE ABL follows a star-network topology in which all devices are connected to a central unmanaged local area network and assigned individual static IPv4 addresses for internal communication. The primary communication protocol within the TRIPLE ABL is MQTT (Message Queuing Telemetry Transport). Each subsystem is identified within the MQTT environment through a unique device identifier, enabling automatic registration and communication with the internal broker. Individual devices operate using custom software stacks written in Python or MicroPython, depending on the hardware platform deployed, including Raspberry Pi 4, Raspberry Pi Zero, and ESP32-S3 microcontrollers. These software layers expose device-specific functions that can be executed remotely through MQTT-based command exchange.

Sample processing and analytical operation within the TRIPLE ABL are designed to be largely automated, with manual intervention currently limited to insertion of the Sterivex™ filter containing the sample. Nevertheless, operators can access and modify system behavior through a graphical user interface when operational adjustments are required. The analytical workflow of the TRIPLE ABL is coordinated by a dedicated orchestration unit implementing a finite-state-machine (FSM) control architecture. Within this framework, the system operates in discrete operational states and transitions between them only when predefined events or conditions are satisfied (Figure 3). Under the current workflow, complete processing of one Sterivex™ sample is expected to require approximately 48 h.

Sample acquisition, handling, and preprocessing

Environmental (saltwater) samples analyzed by the TRIPLE ABL are collected using an aquatic mobile robotic unit, the so-called nanoAUV, developed and constructed by a collaborating research group within the TRIPLE LifeDetect project (Waldmann and Funke, 2020). The nanoAUV utilizes the Sterivex™ filter as the primary sample collection container. The filter cartridge contains a 0.22 μm polyvinylidene fluoride (PVDF) membrane and has an internal hold-up volume of approximately 2.2 mL, enabling capture and retention of microbial particles within the enclosed filter housing.

After the filter unit is docked into the TRIPLE ABL sampling interface, a syringe pump with a stepper motor (Nanotec Electronic GmbH, Captive Linearaktuator–NEMA 17) injects 10 mL isotonic saline solution in reverse direction through the Sterivex™. This step is followed by sterile-air flushing, yielding a total working sample volume of approximately 12.2 mL. The system distinguishes between the external sample volume, defined by the amount of environmental water filtered upstream through the Sterivex™ membrane, and the internal working volume, defined by the eluted volume subsequently processed within the TRIPLE ABL analytical pipeline. In this configuration, the Sterivex™ unit can concentrate biomass from milliliter-to liter-scale environmental samples, whereas the downstream analytical pipeline processes approximately 12 mL of eluted working sample. Simultaneously, a vibration actuator induces vibrations within the filter housing and membrane. Together, the reverse fluid flow and induced shear forces facilitate detachment of particles adhered to the membrane and transport them into the analytical pipeline. This approach enables recovery of intact and potentially viable cells without the use of organic solvents or chemical lysis procedures. The fluidic architecture is organized in a “daisy-chain” topology in which the sample sequentially passes through non-destructive analytical modules before being divided for downstream destructive analyses.

The eluted sample is transferred from the Sterivex™ filter unit to the laboratory compartment through the reverse-flush system using medical-grade silicone tubing (inner diameter: 1.5 mm; outer diameter: 3 mm; VWR Silicone Tubing 228–1,450).

The multimodal microscopy pipeline

Unlike most conventional laboratory microscopes that rely on static glass-slide preparations, the TRIPLE ABL microscopy module employs continuous-flow chambers for automated sample handling and imaging. Precision pinch valves (ASCO Numatics SIRAI S. r.l. S1040900E30D2A) and fixed-volume 25 μL dosing pumps (Lee Hydraulische Miniaturkomponenten GmbH LPLA1251625L) regulate fluid movement and position the liquid column reproducibly within the microscopic field of view. The system integrates three complementary optical modalities, operating at 20× magnification for the dark-field/bright-field microscope, 10x for the fluorescence microscope and 40x for the scanning bright-field microscope and coupled to CMOS-based imaging sensors: (i) dark-field microscopy for visualization of weakly scattering or transparent structures; (ii) bright-field microscopy for general morphological imaging, particle detection, and approximate cell quantification; and (iii) fluorescence microscopy equipped with single wavelength LED excitation sources and optical long-pass filters for detection of potential autofluorescence signals.

The hybrid dark-field/bright-field microscopy unit (Figure 4) is built around a mechanically robust 30 mm × 30 mm aluminum core frame incorporating a 3D-printed flow-chamber holder with integrated illumination LEDs for both bright-field and dark-field operation. The system additionally includes a manually movable focusing assembly containing the camera, tube lens, and 20× microscope objective. Structural holders and mounting components are additively manufactured from polylactic acid (PLA) and directly attached to the aluminum core structure.

The combination of dark-field scattering contrast and bright-field morphological imaging enables label-free visualization and characterization of microorganisms and suspended particles without staining or chemical modification of the sample. Image acquisition is performed using a Raspberry Pi HQ Camera (12.3 MP, IMX477 sensor) controlled by a Raspberry Pi Zero (Raspberry Pi Ltd., 2026). Images are acquired at a resolution of 2048 × 2048 pixels. Although the camera hardware supports video acquisition rates of up to 40 fps, operational frame rates are typically reduced to approximately one fps to balance analytical requirements, data throughput, and power consumption. This configuration nevertheless enables temporally resolved imaging for motility tracking and basic behavioral observation.

The multimodal microscopy pipeline is integrated into the TRIPLE ABL network via MQTT and connected directly to the microfluidic sample-distribution system. When the microscopy pipeline is activated, the orchestration unit instructs the microfluidic subsystem to transfer the sample into the microscope flow chamber. The microscopy modules are then triggered to acquire image sequences. Typically, 120 images are recorded at a frame rate of 1 Hz using a Raspberry Pi HQ camera, and the resulting datasets are stored directly on the internal TRIPLE ABL network-attached storage (NAS) system. The image-processing pipeline consists of grayscale conversion, median denoising (kernel size = 3), background subtraction with negative-value clipping, percentile normalization, and Laplacian sharpening. Processed images can subsequently be analyzed using a motility-detection and tracking workflow to lower human interaction with the analysis of the samples.

The transmitted-light and fluorescence microscopy platforms (Figure 5) are designed as compact, modular imaging units for automated operation inside the TRIPLE ABL laboratory compartment. Both systems are built into a mechanically rigid 20 mm ×20 mm aluminum frame structure, which provides defined mounting interfaces for the camera, objective, illumination source, flow chamber, and positioning components. The two microscopy platforms share the same Z-axis focusing mechanism, allowing the camera–objective assembly to be adjusted reproducibly relative to the sample plane and optical axis. This common focusing architecture reduces mechanical complexity and supports consistent image acquisition across both imaging modalities.

The transmitted-light module is equipped with slide-scan capability and provides contextual bright-field imaging for particle localization, focusing, and sample overview. For this purpose, the flow chamber is mounted on an X–Y positioning stage, enabling raster-based scanning of defined sample regions within the flow chamber. This allows larger sample areas to be imaged sequentially and subsequently reconstructed or evaluated as spatially resolved image datasets. Image acquisition is performed using a Raspberry Pi High Quality Camera equipped with a Sony IMX477R back-illuminated 12.3 MP sensor, a 7.9 mm sensor diagonal, and a pixel size of 1.55 µm × 1.55 µm.

The fluorescence microscopy module is designed for the detection of autofluorescent structures, particularly chlorophyll-associated signals. To support sample positioning and focusing before fluorescence acquisition, the module integrates a red transmitted-light LED for non-fluorescence imaging. After the region of interest has been positioned and focused, the illumination is switched to blue LED excitation to evaluate autofluorescence signals using the corresponding optical emission filtering. Image acquisition in the fluorescence module is performed using a Raspberry Pi Camera Module 2 NoIR. This camera is based on the Sony IMX219 8 MP sensor with a resolution of 3,280 × 2,464 pixels and does not include an infrared-cut filter.

The software workflow supports reproducible image quality by implementing an autofocus routine on the Z-axis. The mechanical design allows the optical components to be integrated into the constrained TRIPLE ABL installation space while maintaining accessibility for adjustment, replacement, and further system development. This modular layout supports the use of microscopy as a non-destructive analytical step before the sample is transferred to downstream analytical modules.

The autofluorescence spectroscopy pipeline

The TRIPLE ABL fluorescence spectroscopy module (Figure 6) is designed to detect optical signatures potentially associated with organic and biological material. The system employs four excitation wavelengths: 265 nm and 275 nm, targeting aromatic amino acids and protein-associated fluorophores; 430 nm, targeting humic- and fulvic-like fluorophores; and 545 nm, targeting pigments such as phycoerythrin and chlorophyll-associated compounds (Coble, 1996). A motorized filter wheel positioned between the cuvette and detector incorporates long-pass filters (280 nm and 455 nm) to suppress excitation-light scattering and isolate fluorescence emission signals.

Spectral detection is performed using an Ibsen Freedom UV-VIS spectrometer covering a wavelength range of 190–850 nm with an optical resolution of approximately 1.7 nm. The detector system is based on a Hamamatsu S11639 CMOS sensor optimized for low-noise UV-VIS acquisition. Signal processing and digitization are performed using an Ibsen DISB-101 interface board, which converts analog detector signals into 16-bit digital output for subsequent data handling and transmission.

The spectrometer is controlled by a Raspberry Pi 4 as a control interface connecting the spectrometer to the ABL’s MQTT network. The fluorescence spectroscopy subsystem can support characterization of chromophoric dissolved organic matter (CDOM), a chemically heterogeneous mixture of dissolved organic compounds with strong absorption in the ultraviolet spectral range (Zeng et al., 2023). Emission spectra from four excitation LED sources obtained from the system can subsequently be analyzed using parallel factor analysis (PARAFAC) or related multivariate decomposition approaches, enabling identification of spectrally distinct components and facilitating improved classification and interpretation of complex samples (Lyu et al., 2021).

A monolithic Hellma 3-in-1 quartz flow-through cuvette (10 × 10 mm 2) is used as the sample cell. The four polished optical windows enable dual-side excitation and 90° fluorescence detection geometry, optionally enhanced using a reflective rear mirror. Figure 6 illustrates the complete fluorescence spectroscopy assembly, including excitation, filtering, detection, thermal regulation, and sample-handling components.

For system control and regulation an NXP LPC1549 microcontroller is used. The processor operates at frequencies up to 72 MHz and includes 256 kB flash memory, 36 kB RAM, and 4 kB EEPROM non-volatile memory. The system normally operates in standby mode, in which command reception remains active while measurement functions remain inactive until a wake-up command is received. Upon entering operational mode, the spectrometer housekeeping- and status data are continuously transmitted. When a measurement command containing the acquisition parameters is received, the cuvette is first refilled with fresh sample material. The acquisition sequence begins with recording of a dark spectrum used for subtraction of detector dark-current contributions from subsequent measurements. Fluorescence spectra are then sequentially acquired for each activated excitation LED until all selected excitation channels have been measured successfully. Following completion of the acquisition cycle, the resulting datasets are transmitted to the central TRIPLE ABL data-management system.

The mass spectrometry pipeline

As described above, in the TRIPLE ABL configuration developed for the Neumayer Station III (2027) mission, the mass spectrometry unit (ABL 01.2–0) is housed in a separate ruggedized Pelican case. Following completion of the analytical routines in the main analytical unit (ABL 01.1–0), approximately 80% of the processed sample volume is routed to the mass spectrometry module (Ecosys-P ESS Ltd).

Because conventional liquid injection can be challenging under Antarctic field conditions, the ABL incorporates a custom-designed headspace-analysis component. In this approach, the aqueous sample is gently heated within a sealed chamber, promoting partitioning of volatile organic compounds (VOCs) and dissolved gases into the gas phase according to Henry’s law. The resulting headspace is subsequently analyzed by the mass spectrometer in order to characterize volatile molecular components, CHNOPS-associated compounds, and potential indicators of chemical disequilibrium that may be relevant to biological activity, including altered methane-to-ammonia relationships (Sellam et al., 2025).

The TRIPLE ABL metagenomics module (optional)

The remaining approximately 20% of the processed sample is reserved for metagenomic analysis performed within an enclosed laboratory-style workspace. At the current development stage of the TRIPLE ABL, DNA extraction has not yet been fully automated and is performed using a modified protocol based on the Qiagen DNeasy PowerWater kit. To make the workflow compatible with compact field-laboratory conditions, centrifugation steps were extended for use with a lower-power centrifuge, and vacuum-based filtration was replaced by syringe-filter-based sample concentration (Pillay et al., 2024; Bian et al., 2026). Ongoing optimization focuses on improving DNA recovery from low-biomass aqueous samples while minimizing contamination risk and preserving workflow robustness.

Library preparation is performed using the Oxford Nanopore Technologies Rapid Barcoding Kit 24 V14 because of its short preparation time (approximately 60 min), moderate DNA input requirements (200 ng DNA in ≤10 μL), and limited laboratory infrastructure requirements. Sequencing is conducted using a compact ONT MinION Mk1B device connected to a laptop computer (Figure 7).

The minimal laboratory equipment used for sample processing includes a syringe and pre-sterilized reusable syringe filter for membrane access and water removal from the backflushed sample, a Bento Lab Pro platform providing compact centrifugation and thermocycling capability, a small vortex mixer used for bead-assisted cell lysis, a compact heating device for reagent preparation, and an Eppendorf BioPhotometer for DNA concentration measurements (Chang et al., 2020). A Qubit fluorometer could alternatively be employed to improve sensitivity and quantitative accuracy for low-concentration DNA samples.

Measurement data handling and transfer

High-volume data storage within the TRIPLE ABL is implemented using a network-attached storage (NAS) system based on a compact low-power computing platform. The selected unit provides high-speed LPDDR5 memory (16 GB RAM) together with substantial storage expandability through six M.2 SSD slots. This architecture is designed to securely store the large raw-data volumes generated by the microscopy modules, fluorescence spectrometer, housekeeping systems, and associated operational log files (Figure 8).

TRIPLE ABL contamination control: main concepts

TRIPLE ABL is designed and operated as a contamination-sensitive closed analytical platform, reflecting the stringent requirements associated with in situ life-detection investigations in extreme environments. Because even trace quantities of exogenous DNA, microorganisms, or chemical residues may produce false-positive biosignature signals or bias downstream analytical workflows, including fluorescence spectroscopy, microscopy, mass spectrometry, and sequencing, contamination control is implemented as a multilayered strategy encompassing both hardware architecture and operational procedures (National Aeronautics and Space Administration, 2021).

All wetted components within the sample pathway, including microfluidic channels, sample chambers, and filter interfaces, are manufactured from chemically inert and abrasion-resistant materials such as selected polymers, stainless steel, and glass that are compatible with validated decontamination procedures. The instrument architecture maintains strict physical separation between a sealed “science section,” inaccessible during field operation, and a separate “housekeeping section” allowing controlled operator access. The TRIPLE ABL housing is fully enclosed to minimize exposure to environmental contaminants, including dust, aerosols, and operator-derived particulates. In addition, sample transfer and handling are conducted under controlled conditions using clean working surfaces and appropriate personal protective equipment (PPE) to reduce cross-contamination risk (Dworkin, 2018; Rettberg, 2019).

To further mitigate contamination arising from internal residues, reagents, and operator interaction, standardized cleaning, flushing, and validation procedures are performed before and during Antarctic deployment. The microfluidic subsystem incorporates automated cleaning cycles between analytical runs using sterile and quality-controlled process fluids to minimize carryover effects and biofilm formation. Specifically, the wetted fluidic path is flushed with 7.5% hydrogen peroxide (H 2 O 2) to reduce residual organic and nucleic-acid contamination, followed by rinsing with ultrapure, molecular-grade DNA-free water. This chemical cleaning cycle is complemented by UV-C irradiation of the internal laboratory volume between sample runs. External surfaces and critical interfaces, including sample inlets, are regularly decontaminated according to a standardized wipe protocol before and after field operation.

Potential contamination sources, including residual material within fluidic lines, contaminated reagents, aerosol backflow from waste compartments, and direct human contact, are systematically addressed through preventive and corrective measures. These include routine system inspections, restricted access to critical components, and implementation of blank-control measurements for background monitoring (Debus and Viso, 2002). In cases of suspected contamination, cleaning cycles are repeated, consumables are replaced, and the system may be temporarily removed from operation for detailed inspection and decontamination. Together, these measures are intended to ensure a high level of cleanliness, traceability, and analytical reliability, supporting robust interpretation of biosignature-related measurements under field conditions consistent with planetary-protection principles (Committee on Space Research, 2021).

Validation strategy and test campaign

The ABL development program follows a staged verification and validation strategy based on a four-tier model philosophy consisting of a Breadboard Model (BB-ABL), Engineering Model (EM-ABL), Qualification Model (QM-ABL), and Field-deployable Model (FM-ABL), with the QM-ABL subsequently refurbished into the FM-ABL following qualification testing.

During the development phase, BB-ABL and EM-ABL units are used to characterize the individual analytical pipelines (fluorescence spectroscopy, fluorescence microscopy, and mass spectrometry) using laboratory-prepared positive and negative controls together with field-representative reference standards. Subsystem-level verification additionally addresses fluidic distribution, power-management functionality, and housekeeping electronics.

Qualification testing of the integrated QM-ABL includes end-to-end validation of analytical workflows across all four analytical modalities, assessment of cross-contamination and decontamination performance using alternating positive and blank sample series, climate-chamber testing across the operational temperature range of −25 °C to +40 °C, and verification of the overall system-level power budget. Final acceptance testing of the FM-ABL is planned during the Antarctic field campaign on the shelf-ice test site, where operational robustness, analytical performance, data quality, and decontamination effectiveness will be evaluated under representative environmental conditions.

Thermal homeostasis of the TRIPLE ABL

The thermal-management performance of the TRIPLE ABL system was evaluated in an environmental chamber at 8 °C to characterize temperature stability, thermal gradients, and power consumption under controlled conditions (Figure 9). Although these measurements were performed above the lowest anticipated field temperatures and using a dummy ABL box, they provide an initial basis for estimating system behavior under more extreme deployment conditions through thermal modeling and cautious extrapolation. Future validation tests are planned for sub-zero conditions before the Antarctic deployment.

Sample recovery from the Sterivex™ system

Laboratory-scale validation experiments indicated that the developed backflush-based recovery system can detach microorganisms and particulate material from filtration membranes and transfer them into downstream analytical workflows within the TRIPLE ABL architecture. These findings support the underlying hypothesis that a combination of hydrodynamic backflushing and mechanical stimulation can partially overcome adhesive interactions between microbial cells, particulate matter, and membrane filter surfaces.

At the same time, microbial recovery proved to be strongly condition-dependent and was influenced by sample composition, particulate load, and the formation of fouling layers or early-stage biofilms. Under such conditions, complete detachment of all retained microorganisms cannot be expected, because cell adhesion and particle-associated binding mechanisms remain partially resistant to purely hydraulic or mechanical removal processes. Recovery efficiency should therefore be regarded as a dynamic parameter governed by physicochemical boundary conditions rather than as a fixed intrinsic system property. While the qualitative feasibility of the approach was clearly demonstrated, the limited experimental dataset and inherent variability of environmental samples currently restrict robust quantitative evaluation. Indeed, from a technological perspective, the experiments revealed several engineering constraints and optimization pathways relevant for future TRIPLE ABL development.

Increased sediment load resulted in elevated hydraulic resistance within the filtration system, directly affecting flow stability, mechanical stress on fluidic components, and overall process reproducibility. Transition from peristaltic pumping to syringe-pump-based fluid handling improved volumetric precision but simultaneously altered flow characteristics by reducing pulsatile shear forces that may contribute to enhanced particle detachment. This trade-off highlights the importance of coupling controlled fluid dynamics with mechanical stimulation to optimize recovery efficiency. Future work should therefore systematically investigate the interactions between flow profiles, pressure regimes, and mechanical excitation, ideally supported by real-time sensor feedback and adaptive process control. Some promising approaches include investigation of osmotic effects, for example, through hypertonic treatment conditions, on cell–surface interactions relevant to subglacial and other icy-world analogue environments.

TRIPLE ABL microscopy data

The dark-field/bright-field microscopy unit, one of three optical modalities integrated into the TRIPLE ABL, was validated using an environmental coastal water sample collected near Noordwijk, Netherlands (Figure 10).

Bright-field imaging (A) provides general morphological contrast across the full dynamic range of the sample, while dark-field imaging (B) reveals weakly scattering and largely transparent microorganisms that are poorly visible in bright-field, effectively extending the detectable dynamic range of the system. Both modalities operate at 20× magnification with an image resolution of 2048 × 2048 pixels, yielding sufficient spatial resolution for detection and characterization of microorganisms in the low micrometer size range, including microalgae, cyanobacteria, and diatoms. Residual stray light is visible in the dark-field images, which will be addressed in future optical refinements. The processed dark-field image (C), derived from (B) through the cleanup pipeline, demonstrates enhanced particle contrast suitable for downstream segmentation and analysis. The resulting image data from both modalities are suitable for downstream automated image analysis, including morphological classification, particle segmentation, and motility detection, providing assumption-agnostic biosignature indicators independent of biochemical composition.

The fluorescence microscopy module was validated using the same environmental coastal water sample collected near Noordwijk, Netherlands (Figures 10D–F). Red LED context illumination (E) provides spatial orientation and general particle distribution within the sample, while blue LED excitation (465 nm) with a 650 nm long-pass filter reveals chlorophyll-containing microorganisms via autofluorescence (D), indicated by the arrows. The detected autofluorescence signals confirm the presence of photosynthetically active microorganisms in the sample, demonstrating the capability of the module to identify chlorophyll-containing organisms without staining or chemical modification. Image (F) shows the same sample region acquired with a laboratory phase contrast microscope Axiovert 25 CFL (Carl Zeiss Co.), provided as a morphological reference.

This experiment was not designed as a quantitative ecological survey, but as a qualitative feasibility test to verify whether the automated TRIPLE ABL microscopy module can generate spatially consistent and complementary image information across multiple optical modalities from the same environmental sample region.

Fluorescence spectroscopy data

Figure 11 presents an example of fluorescence spectral response of key metabolic cofactors and whole-cell microbial samples acquired using the TRIPLE ABL fluorescence spectroscopy module. Panel (A) illustrates the excitation-dependent spectral behavior of NADH and FAD standards, whereas panel (B) shows characteristic fluorescence fingerprints of representative microorganisms obtained under dual-wavelength excitation conditions. Together, these data demonstrate the capability of the system to resolve both molecular- and cellular-scale optical biosignatures.

The spectra obtained from NADH/FAD mixtures exhibited characteristic emission maxima consistent with established photophysical properties, particularly the flavin-associated emission band in the 500–550 nm range under blue-light excitation, indicating appropriate operational performance of the spectroscopy module (Müllerová et al., 2022). The microbial spectra additionally displayed distinct and reproducible signatures consisting of both fluorescence-emission and scattering components that may enable differentiation between bacterial and yeast samples, in general agreement with previously reported microbial autofluorescence fingerprints (Pan et al., 2008; Himics et al., 2025; Larionov et al., 2026). At the same time, microbial autofluorescence spectra are known to depend strongly on both biological and instrumental factors, including growth phase, metabolic state, nutrient availability, environmental stress conditions, excitation wavelength, detector sensitivity, spectral filtering, and acquisition geometry (Croce and Bottiroli, 2014; Angelotti de Ponte Rodrigues, Natália et al., 2024; Case et al., 2024). Consequently, substantial inter-study variability has been reported even for identical microbial species (Arabi et al., 2026).

Environmental samples collected under Antarctic or ocean-world analogue conditions are frequently characterized by extremely low biomass and highly diluted fluorophore concentrations. As a result, fluorescence signals may approach or fall below practical detection limits, complicating reliable identification of target compounds (Zhong et al., 2018; D’Sa et al., 2021). In addition, residual particulate material introduced into the cuvette during reverse flushing may contribute to inner-filter effects and fluorescence reabsorption artifacts. To address these limitations, the current development phase focuses on systematic determination of detection limits and signal-to-noise characteristics of the TRIPLE ABL fluorescence spectroscopy module.

In parallel, a reference database of environmental seawater fluorescence spectra is being established using both natural samples and laboratory-cultivated microbial systems. This database is intended to support future identification, comparison, and interpretation of fluorescence signatures in unknown environmental samples.

Pilot mass-spectrometry measurements

Initial functional verification of the mass-spectrometry channel was performed at breadboard level using the ESS EcoSys-P portable mass spectrometer in a temperature-controlled laboratory at FH Aachen, at approximately 22 °C. The instrument was operated over its nominal 1–200 amu mass range across 100 consecutive scan cycles. Prior to environmental-sample testing, the channel was evaluated using argon, nitrogen, and carbon dioxide reference gases. These measurements reproduced the expected peaks at their nominal m/z values and confirmed correct species assignment across the low-to mid-mass range. For an integrated end-to-end test of the headspace interface, a 3.5% NaCl aqueous matrix was subsequently analyzed, and acetone was introduced part-way through the run as a volatile test analyte.

Following vacuum stabilization and ion-source warm-up, a baseline scan was acquired using a clean inlet to assess background signal levels and potential spurious peaks. Sample-derived spectra were then recorded continuously while peak position (m/z), peak intensity, signal-to-noise ratio, and baseline drift were monitored. After linear mass calibration anchored to H 2 O, N 2, O 2, and CO 2, with residual mass deviation below 0.2 amu, the spectra reproduced the expected water- and atmosphere-derived peaks at m/z 18, 28, 32, and 44, respectively (Figure 12). Signal-to-noise ratios exceeded the predefined threshold of 10 by several orders of magnitude, reaching approximately ×5 10 5 at m/z 28 and 1.6 × 10 5 at m/z 32 relative to a high-mass baseline noise of approximately 2 × 10 −12 A.

Upon acetone introduction, characteristic acetone-related fragments at m/z 43 and 58 emerged clearly above the baseline, with an estimated S/N of approximately 1 × 10 4 at m/z 43. This confirmed that the headspace channel can resolve an introduced volatile analyte against an aqueous saline matrix and can track compositional changes across successive scan cycles (Figure 12B). The high-mass region remained close to the noise floor and did not show prominent additional peaks, indicating stable baseline behavior under the tested conditions. As expected for a headspace-based method, non-volatile tyrosine present in the matrix did not produce a detectable signal. This result delineates the current mass-spectrometry channel as primarily sensitive to volatile compounds and dissolved gases, rather than to non-volatile biomolecules retained in the aqueous phase.

Metagenomic module: current status and outlook

At the present development stage, the metagenomic module is retained as an additional, analyst-operated capability rather than as a validated in-line analytical stage. DNA extraction, library preparation, and MinION flow-cell handling are not yet automated, and several constraints currently preclude full integration into the closed fluidic system. Mechanical lysis by bead beating or vortexing is difficult to reconcile with vibration-sensitive optical and mass-spectrometric instruments; wet-chemistry reagents introduce contamination backgrounds that must be characterized and controlled; and nanopore flow cells require controlled storage conditions that are difficult to guarantee during extended field campaigns. Purely fluidic extraction strategies, enzyme-based lysis, low-pipetting library-preparation chemistries, and emerging solid-state nanopore technologies may provide routes toward future automation, but these approaches were beyond the scope of the present prototype. Sequencing is therefore retained in the TRIPLE ABL architecture as a downstream, analyst-gated option for samples flagged by the non-destructive screening stages. Pilot sequencing data are not reported here as a performance claim. For future implementations of the DNA module scientists are keenly observing solid state nanopores and their development (Dekker, 2007).

Relevance to ocean-world sampling scenarios. limitations and outlook

Conceptually, the TRIPLE ABL is designed to contribute to progressively higher levels of confidence within the “Ladder of Life Detection” framework (Neveu et al., 2018). Because life is a highly complex and heterogeneous phenomenon, definitive demonstration of biological activity is unlikely to be achievable using a single analytical instrument alone (Malaterre et al., 2023). The central scientific objective of the TRIPLE ABL concept is therefore the integration of complementary multimodal analytical approaches capable of probing different classes of potential biosignatures.

Within this framework, microscopy contributes morphological and behavioral observations, including detection of cell-like structures, particulate organization, and potential non-Brownian motility patterns. Fluorescence spectroscopy provides optical and biochemical information through detection of autofluorescence signatures potentially associated with biologically relevant fluorophores, including pigments and dissolved organic compounds. Mass spectrometry contributes elemental and molecular information relevant to environmental habitability and chemical disequilibrium, including characterization of CHNOPS-associated compounds and volatile chemical species. Finally, metagenomic analysis enables characterization of DNA- or RNA-based biological systems through assessment of genetic diversity together with potential taxonomic, metabolic, and ecological features. By combining these complementary evidence layers within a unified analytical workflow, the ABL concept aims to move beyond isolated “detection” events toward more robust characterization and contextual interpretation of candidate biosignatures in ocean-world analogue environments.

As a terrestrial testbed, the current TRIPLE ABL was not optimized for launch mass or volume; its modular architecture is, however, intended to permit subsequent subsystem-level miniaturization—for example, microfluidic integration of the fluidic path, compact integrated optics, and smaller mass-analyser front-ends—as a prerequisite for any future flight implementation.