Introduction
Mars Sample Return (MSR) is a mission defined as ‘Category V restricted Earth return’ by the Committee on Space Research's (COSPAR) Planetary Protection Policy (https://cosparhq.cnes.fr/scientific-structure/panels/panel-on-planetary-protection-ppp/). To cover the category description element in the policy stating a need to conduct timely analyses of any unsterilized sample collected and returned to Earth, under strict containment, and using the most sensitive techniques, an international working group was organized by COSPAR and the sample safety assessment framework (SSAF) was developed (Kminek et al., Reference Kminek, Benardini, Brenker, Brooks, Burton, Dhaniyala, Dworkin, Fortman, Glamoclija, Grady, Graham, Haruyama, Kieft, Koopmans, McCubbin, Meyer, Mustin, Onstott, Pearce, Pratt, Sephton, Siljeström, Sugahara, Suzuki, Suzuki, Zuilen and Viso2022a).
The SSAF targets living organisms, their resting states (e.g. spores, cysts), or their remains in Martian materials. The tentative level of safety assurance is a risk value of 1 in a million chance of failing to detect life, if it is present. The risk value is estimated by Bayesian statics that takes the likelihood of samples and subsamples containing life and the sensitivity of analytical methods into consideration for the generation of probabilities. To reduce the number and volume of samples to be consumed for planetary protection, a subsampling procedure is employed. The SSAF identified optimal subsampling targets to be regions of rock samples with high concentrations of pore-spaces or fractures. As life is likely to be detected where the prolonged presence of water has formed clays by rock–water interactions, rock samples containing clays are prioritized as subsampling targets. Subsamples are subjected to a test sequence, in which non-destructive analytical steps are followed by destructive analytical steps.
Implementation of the SSAF poses challenges because most of the investigations need to be conducted within biological containment. The SSAF is therefore described at a level of detail that will support planning activities for sample receiving facilities (SRF). Three major open issues were raised to effectively implement and optimize the SSAF, specifically the need to: (1) set a level of assurance to exclude the presence of Martian life in the samples, (2) carry out an analogue test programme, and (3) acquire relevant contamination knowledge from flight and ground elements. In the analogue test programme, analytical steps need to be validated by testing with analogue samples potentially returned from Mars. In the lists of analytical instruments for SRF, Fourier-transform infrared (FT-IR) spectroscopy is requested from both the SSAF and Mars Sample Return Science Planning Group 2 (MSPG2) (Carrier et al., Reference Carrier, Beaty, Hutzler, Smith, Kminek, Meyer, Haltigin, Hays, Agee, Busemann, Cavalazzi, Cockell, Debaille, Glavin, Grady, Hauber, Marty, McCubbin, Pratt, Regberg, Smith, Summons, Swindle, Tait, Tosca, Udry, Usui, Velbel, Wadhwa, Westall and Zorzano2022; Haltigin et al., Reference Haltigin, Hauber, Kminek, Meyer, Agee, Busemann, Carrier, Glavin, Hays and Marty2022; Meyer et al., Reference Meyer, Kminek, Beaty, Carrier, Haltigin, Hays, Agree, Busemann, Cavalazzi, Cockell, Debaille, Glavin, Grady, Hauber, Hutzler, Marty, McCubbin, Pratt, Regberg, Smith, Smith, Summons, Swindle, Tait, Tosca, Udry, Usui, Velbel, Wadhwa, Westall and Zorzano2022; Tait et al., Reference Tait, McCubbin, Smith, Agee, Busemann, Cavalazzi, Debaille, Hutzler, Usui and Kminek2022; Tosca et al., Reference Tosca, Agee, Cockell, Glavin, Hutzler, Marty, McCubbin, Regberg, Velbel and Kminek2022; Velbel et al., Reference Velbel, Cockell, Glavin, Marty, Regberg, Smith, Tosca, Wadhwa, Kminek and Meyer2022; Kminek et al., Reference Kminek, Meyer, Beaty, Carrier, Haltigin and Hays2022b). FT-IR spectroscopy has the capability of identifying inorganic and organic materials. However, a spatial resolution of FT-IR microscopy is limited by the diffraction limit of IR light (~10 μm), which hinders the single-cell sensitivity for many microorganisms.
Optical-photothermal infrared (O-PTIR) spectroscopy has a superior spatial resolution (~0.5 μm) to FT-IR microscopy, because light scattering caused by thermal expansion under pulsed IR light with a beam diameter of ~10 μm is detected by a green laser (532 nm) with a beam diameter of ~0.5 μm and a photodiode (Li et al., Reference Li, Zhang, Bai, Wang, Liang and Cheng2019; Lima et al., Reference Lima, Muhamadali, Xu, Kansiz and Goodacre2021). Light-scattering response is monitored during tuning the wavenumber of the IR source, which creates a FT-IR like spectrum. In addition to the spatial superiority, O-PTIR needs no preparation steps required for FT-IR microscopy. In this study, O-PTIR spectroscopy was applied to a rock core sample where the dense colonization of microbes has been demonstrated in clay-filled fractures as an analogue for MSR (Suzuki et al., Reference Suzuki, Yamashita, Kouduka, Ao, Mukai, Mitsunobu, Kagi, D'Hondt, Inagaki and Morono2020).
Methods
Sampling and sample preparations
The rock core sample coded as U1365E-8R4 (109.6 m below the seafloor) was obtained by drilling of the 104-million-year-old basaltic basement during Integrated Ocean Drilling Project (IODP) expedition #329. IODP expedition #329 targeted life beneath the seafloor of the South Pacific Gyre (SPG), where surface photosynthetic activity is exceedingly low (D'hondt et al., Reference D'Hondt, Inagaki, Zarikian, Abrams, Dubois, Engelhardt, Evans, Ferdelman, Gribsholt, Harris, Hoppie, Hyun, Kallmeyer, Kim, Lynch, McKinley, Mitsunobu, Morono, Murray, Pockalny, Sauvage, Shimono, Shiraishi, Smith, Smith-Duque, Spivack, Steinsbu, Suzuki, Szpak, Toffin, Uramoto, Yamaguchi, Zhang, Zhang and Ziebis2015). This ultra-oligotrophic feature favours microorganisms living independently from photosynthetic organics (Morono et al., Reference Morono, Ito, Hoshino, Terada, Hori, Ikehara, D'Hondt and Inagaki2020). This feature appears to be analogous to the Martian surface, because the growth of photosynthetic organisms is likely suppressed under frozen and/or arid conditions (Onstott et al., Reference Onstott, Ehlmann, Sapers, Coleman, Ivarsson, Marlow, Neubeck and Niles2019).
Sample preparations for the rock core sample were previously reported (Sueoka et al., Reference Sueoka, Yamashita, Kouduka and Suzuki2019). After the recovery of the rock core sample, the contaminated exterior of the rock core sample was removed by a sterilized hammer and chisel until fluorescent microspheres present in the drilling fluids were undetected from the interior of the rock core sample by a UV fluorescent microscope. A portion of the rock interior was ground into powder by a sterilized mortar and pestle. A clay-sized fraction was separated by suspending the powder sample in deionized water, from which a fraction larger than the clay-sized fraction was removed by centrifugation at 3000 rpm for 5 min. After the separation, the supernatant was freeze-dried and stored at −30°C. For the preparation of a 100 μm thin section, a fracture-bearing rock piece was embedded in LR White resin (London Resin Co. Ltd., Aldermaston, England) and then thinned by polishing with corundum powder and diamond paste. All laboratory woks were performed in a clean bench with sterilized apparatus and reagents.
Conventional FT-IR microscopy
The clay-sized fraction in the powder sample was mounted on a diamond cell (Diamond EX'Press II, S.T. Japan Inc.). FT-IR spectra from 700 to 4000 cm−1 were obtained from a 10 μm square region by a Shimadzu AIM-9000 FT-IR microscope in combination with an IRTracer-100 (Cassegrain 15 × objective). A transmission mode was used with a spectral resolution of 0.25 cm−1. Clay Science Society of Japan (JCSS) reference clay samples such as montmorillonite JCSS3101 ((M + .97)[Si7.8Al.02][Al3.3Fe-.2Mg.6]O20(OH)4) and saponite JCSS3501 ((M + .98)[Si7.2Al.08][Mg6.0]O20(OH)4) and a reference sample of nontronite coded NAu-2 ((M + .97)[Si7.57Al.01Fe.42][Al.52Fe3.32Mg.7]O20(OH)4 (Keeling et al., Reference Keeling, Raven and Gates2000)) were used as references to fit the spectra of the unknown. In addition, the Attenuated Total Reflection (ATR) mode (Shimadzu ATR objective Ge prism) was used to obtain FT-IR spectra from the nontronite reference. In addition, the 100 μm thick section was mounted on the stage of the microscope. We attempted to obtain FT-IR spectra from areas associated with the clay-filled fractures using the ATR mode.
O-PTIR and Raman spectroscopy
To acquire O-PTIR spectra from the thin section of the rock interior at the submicron resolution, a mIRage infrared microscope (Photothermal Spectroscopy Corp., Santa Barbara, USA) was used in reflectance mode (Cassegrain 40 × objective) with a continuous wave 532 nm laser as probe beam. The pump beam consisting of a tunable QCL device (800–1895 cm−1; 2 cm−1 spectral resolution and 10 scans per spectrum) was used to obtain O-PTIR spectra over the mid-IR ranges. Raman spectra were collected from 4000 to 200 cm−1 with 1 s integration and 20 scans for averaging. For microbiological references, co-cultured cells of Nanoarchaeota strain MJ1 and Metallosphaera sp. strain MJ1HA (JCM33617) and cultured cells of Escherichia coli (NBRC13168) were freeze-dried. The cultured cells, the reference clay samples and a powder of LR White resin were mounted on a CaF2 disk for analysis.
Results
Conventional FT-IR microscopy for the rock core sample
We collected FT-IR spectra from the clay-sized fractions of U1365E-8R4 and smectite references with a conventional FT-IR microscope with the transmission mode (Fig. 1). In smectite spectra from a 10 μm square region, there is a broad peak centred around 3390 cm−1, which is attributed to vibration modes of interlayer H2O (Bishop et al., Reference Bishop, Pieters and Edwards1994; Madejová et al., Reference Madejová, Komadel and Čičel1994). A peak at ~1000 cm−1 is attributed to Si2-O symmetric and asymmetric stretching modes (Ellerbrock et al., Reference Ellerbrock, Stein and Schaller2022). Between 3700 and 3550 cm–1, peak and shoulder features are diagnostic to types of cation-OH stretching modes. The Fe(III)2-OH stretching mode has a peak feature at 3550 cm–1(Gates, Reference Gates and Kloprogge2005), whereas well-defined shoulder features at 3630 and 3680 cm–1 are attributed to the Al2–OH and Mg2–OH stretching modes (Grauby et al., Reference Grauby, Petit, Decarreau and Baronnet1993, Reference Grauby, Petit, Decarreau and Baronnet1994). FT-IR spectra were similar between U1365E-8R4 and nontronite. The ATR mode was used to obtain FT-IR spectra from the nontronite reference under the same conditions for the transmission mode. The FT-IR spectra have low signal-to-noise ratios with a major peak at ~1000 cm−1 shifted towards the higher wavenumber than that obtained from the same reference by the transmission mode (Fig. 1). In addition, a peak attributed to the H2O bending mode at 1635 cm–1 and peaks between 3700 and 3550 cm–1 diagnostic to smectite were absent in the spectra. As for the thin section, FT-IR spectra diagnostic to smectite and microbial cells were not obtained (data not shown). FT-IR spectra for microbial cells were not obtained via the ATR mode presumably because of the limitation spatial resolution.
Figure 1. FT-IR microscopy spectra from smectite references and the clay fraction of the rock core. Peak attributions are described in the text.
O-PTIR spectroscopy for mapping signals from microbes and smectite
For the thin section of the rock core, O-PTIR spectroscopy was used to map the signal intensities of 1000 and 1530 cm−1, at which major peaks of smectites and microbial cells were respectively obtained (Fig. 2(a)–(e)). At the brownish rim of a greenish fracture made of a Fe-rich mica mineral called celadonite (Fig. 2(a)–(c)), both signal intensities were high (Fig. 2(c)–(e), pink and light blue points), indicating the co-occurrence of smectites and microbial cells.
Figure 2. Photographs of a nontronite-bearing fracture in the thin section from the rock core interior (a–c) with increasing magnification. Intensity maps of optical photothermal infrared (O-PTIR) spectra in a region highlighted with a yellow square in Fig. 2c at 1000 cm−1 (d) and 1530 cm−1(e). O-PTIR spectra from duplicate analyses of pink points in Fig.. 2(c)–(e), co-cultured cells of Nanoarchaeota strain MJ1 and Metallosphaera sp. strain MJ1HA (JCM33617) and cultured cells of Escherichia coli (NBRC13168) and LR White resin (f). Peak assignment was based on Movasaghi et al. (Reference Movasaghi, Rehman and Rehman2008) and Ellerbrock et al. (Reference Ellerbrock, Stein and Schaller2022) and references therein.
O-PTIR spectroscopy for diagnostic spectra from microbes
In O-PTIR spectra obtained from loci with the high signal intensity at 1530 cm−1, peaks attributed to amide I and II (an indicator of peptides) were evident, in addition to a peak at 1450 cm−1 attributed to the bending vibration (scissoring) of CH2 groups in lipids and a peak band at 1390 cm−1 attributed to the stretching vibration of COO of amino acid side chains and fatty acids (Lima et al., Reference Lima, Muhamadali, Xu, Kansiz and Goodacre2021). The spectra were similar to microbial cells but distinct from that from LR White resin (Fig. 2f). It should be noted that the sample damage was not evident during the repeated measurements by O-PTIR.
O-PTIR spectroscopy for diagnostic spectra from smectite
In O-PTIR spectra from 800 to 1800 cm−1, Fe(III)-bearing smectites exhibit a pair of bands at 815–817 and 870 cm–1 attributed to the Fe(III)2–OH and the Fe(III)–Al–OH bending modes (Grauby et al., Reference Grauby, Petit, Decarreau and Baronnet1994; Gates, Reference Gates and Kloprogge2005; Andrieux and Petit, Reference Andrieux and Petit2010). In addition, a peak attributed to the H2O bending mode occurs at 1635 cm–1 (Bishop et al., Reference Bishop, Pieters and Edwards1994; Madejová et al., Reference Madejová, Komadel and Čičel1994). From the thin section of U1365E-8R4, an O-PTIR spectrum similar to that of nontronite was obtained at the points with and without the peaks attributed to Amide I and II (Fig. 3), except for a peak attributed to the Fe(III)–Al–OH bending mode at 870 cm–1.
Figure 3. Duplicate optical photothermal infrared (O-PTIR) spectra of light blue points in Fig. 2(c)–(e) and smectite references. Peak attributions are described in the text.
μ-Raman spectroscopy for comparison with O-PTIR spectroscopy
We used μ-Raman spectroscopy with an excitation wavelength of 532 nm to obtain spectra from the same spots (Fig. 4), from which O-PTIR spectra identical to nontronite were obtained (Fig. 3). No obvious peaks were present in the Raman spectra (Fig. 4). Next, Raman spectra were obtained from the bacterial culture and the same spots (Fig. 4), from which O-PTIR spectra including sharp amide peaks were obtained (Fig. 2). No obvious peaks were obtained from the bacterial culture and the spots due to the interference by autofluorescence.
Figure 4. Raman spectra from light blue and pink points shown in Fig 2(c)–(e) and cultured cells of Escherichia coli (NBRC13168).
Discussion
O-PTIR sensitivity for detecting microbial cells and adjacent smectites from an analogue rock sample
On Mars, basaltic lava is ubiquitous, where Fe- and Mg-bearing smectites with compositions ranging from nontronite (iron endmember) to saponite (magnesium endmember) are the most common clay minerals formed by silicate weathering or hydrothermal alteration (Mustard et al., Reference Mustard, Murchie, Pelkey, Ehlmann, Milliken, Grant, Bibring, Poulet, Bishop, Dobrea, Roach, Seelos, Arvidson, Wiseman, Green, Hash, Humm, Malaret, McGovern, Seelos, Clancy, Clark, Marais, Izenberg, Knudson, Langevin, Martin, McGuire, Morris, Robinson, Roush, Smith, Swayze, Taylor, Titus and Wolff2008; Ehlmann et al., Reference Ehlmann, Mustard, Murchie, Bibring, Meunier, Fraeman and Langevin2011). Fe, Mg-rich smectites are similarly observed in settings associated with basaltic lava on Earth (Alt, Reference Alt1988; Teagle et al., Reference Teagle, Alt, Bach, Halliday and Erzinger1996; Yamashita et al., Reference Yamashita, Mukai, Tomioka, Kagi and Suzuki2019). We regard the rock core sample drilled from ancient basaltic lava as an analogue rock sample, given that basaltic rock fractures are filled with nontronite.
In our previous studies, a conventional X-ray diffraction analysis was performed using clay-sized fractions separated from crushed rock samples (Sueoka et al., Reference Sueoka, Yamashita, Kouduka and Suzuki2019; Yamashita et al., Reference Yamashita, Mukai, Tomioka, Kagi and Suzuki2019). In addition, ~100 μm thick sections made from the same core samples by embedding the fracture-bearing rock piece in hydrophilic resin called LR White were subjected to clay mineral characterization using scanning electron microscopy coupled with energy-dispersive spectroscopy (SEM-EDS) and μ-Raman spectroscopy. From regions in the thin section with EDS spectra similar to nontronite, no peaks were obtained in Raman spectra in our previous study (Yamashita et al., Reference Yamashita, Mukai, Tomioka, Kagi and Suzuki2019). Thus, transmission electron microscopy equipped with EDS (TEM-EDS) analysis was necessary for the nanoscale mineralogical identification of nontronite.
Greenish signals from microbial cells stained with SYBR-Green I in the thin sections were visualized by fluorescence microscopy before SEM-EDS analysis was performed for the clay mineral characterization (Suzuki et al., Reference Suzuki, Yamashita, Kouduka, Ao, Mukai, Mitsunobu, Kagi, D'Hondt, Inagaki and Morono2020). Then, microbial cells in the thin sections were characterized by μ-Raman spectroscopy (Suzuki et al., Reference Suzuki, Yamashita, Kouduka, Ao, Mukai, Mitsunobu, Kagi, D'Hondt, Inagaki and Morono2020) (Fig. 5). However, fluorescence signals obscured peaks from Raman shifts from the DNA-stained microbial cells (Suzuki et al., Reference Suzuki, Yamashita, Kouduka, Ao, Mukai, Mitsunobu, Kagi, D'Hondt, Inagaki and Morono2020). To confirm if the greenish signals are originated from microbial cells, nanoscale secondary ion mass spectrometry (NanoSIMS) was used for submicron-scale mapping of carbon, nitrogen, sulphur and phosphorous.
Figure 5. Comparison of analytical data between NanoSIMS (upper) and O-PTIR (lower) (a) and between TEM-EDS (upper) and O-PITR (lower) (b). Rock characterization procedures performed in our previous and present studies (c). NanoSIMS and TEM-EDS data are modified from Suzuki et al. (Reference Suzuki, Yamashita, Kouduka, Ao, Mukai, Mitsunobu, Kagi, D'Hondt, Inagaki and Morono2020) and Yamashita et al. (Reference Yamashita, Mukai, Tomioka, Kagi and Suzuki2019), respectively. SEM, scanning electron microscopy; EDS, energy-dispersive x-ray spectroscopy; FIB, focused ion beam; TEM, transmission electron microscopy; NanoSIMS, nanoscale secondary ion mass spectrometry; O-PTIR, optical-photothermal infrared.
However, it was necessary to fabricate thin sections with a thickness of ~3 μm using a focused ion beam (FIB). To identify minerals around microbial cells, FIB sections needed to be thinned down to a thickness of 100 nm for TEM-EDS. The higher the spatial resolution, the more damaged the sample. Nevertheless, high-resolution analytical data were crucial to determine mineral identity and the biogenicity of signals from SYBR-Green I.
From the rock fracture, we obtained the O-PTIR spectra identical to those from microbial cultures (Fig. 2). These results are consistent with those obtained by NanoSIMS analysis of the FIB-fabricated section (Fig. 5a). The presence of a Fe(III)-Al-OH bend in the O-PTIR spectra adjacent to those of microbial cells is also consistent with TEM-EDS data showing the high Al content in nontronite in the rock fracture (Yamashita et al., Reference Yamashita, Mukai, Tomioka, Kagi and Suzuki2019). The in-situ capability of O-PTIR spectroscopy for identifying smectites is also comparable to the high-resolution analyses by TEM-EDS (Fig. 5b). Thus, O-PTIR spectroscopy can be used for detecting microbial cells and smectite, without using our previous published procedures (Fig. 5c). TEM and NanoSIMS are used to resolve cellular ultrastructures (Wanger et al., Reference Wanger, Moser, Hay, Myneni, Onstott and Southam2012) and to provide ppm-level elemental abundances and isotopic ratios (Ito and Messenger, Reference Ito and Messenger2008, Reference Ito and Messenger2016), respectively. Therefore, O-PTIR is a good complimentary technique but not a total substitute.
Considerations for the usage of O-PTIR spectroscopy in SRF
Analytical instruments required by sample safety assessment and basic characterization, preliminary examination and time- and sterilization-sensitive sciences are environmental SEM and deep ultraviolet (DUV) fluorescence microscopy/μ-Raman spectroscopy (Carrier et al., Reference Carrier, Beaty, Hutzler, Smith, Kminek, Meyer, Haltigin, Hays, Agee, Busemann, Cavalazzi, Cockell, Debaille, Glavin, Grady, Hauber, Marty, McCubbin, Pratt, Regberg, Smith, Summons, Swindle, Tait, Tosca, Udry, Usui, Velbel, Wadhwa, Westall and Zorzano2022; Tait et al., Reference Tait, McCubbin, Smith, Agee, Busemann, Cavalazzi, Debaille, Hutzler, Usui and Kminek2022; Tosca et al., Reference Tosca, Agee, Cockell, Glavin, Hutzler, Marty, McCubbin, Regberg, Velbel and Kminek2022; Velbel et al., Reference Velbel, Cockell, Glavin, Marty, Regberg, Smith, Tosca, Wadhwa, Kminek and Meyer2022), partly because these analytical instruments are non- or minimally destructive without any sample preparation prior to the analyses. The Mars 2020 Perseverance rover is currently investigating the Noachian Jezero crater, where fine-grained basaltic igneous rocks within the Máaz and Séítah formations were analysed by SHERLOC (Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals), a DUV fluorescence/Raman spectrometer (Scheller et al., Reference Scheller, Razzell Hollis, Cardarelli, Steele, Beegle, Bhartia, Conrad, Uckert, Sharma, Ehlmann, Abbey, Asher, Benison, Berger, Beyssac, Bleefeld, Bosak, Brown, Burton, Bykov, Cloutis, Fairén, DeFlores, Farley, Fey, Fornaro, Cox, Fries, Hickman-Lewis, Hug, Huggett, Imbeah, Jakubek, Kah, Kelemen, Kennedy, Kizovski, Lee, Liu, Mandon, McCubbin, Moore, Nixon, Nuñez, Rodriguez Sanchez-Vahamonde, Roppel, Schulte, Sephton, Sharma, Siljestrom, Shkolyar, Shuster, Simon, Smith, Stack, Steadman, Weiss, Werynski, Williams, Wiens, Williford, Winchell, Wogsland, Yanchilina, Yingling and Zorzano2022; Corpolongo et al., Reference Corpolongo, Jakubek, Burton, Brown, Yanchilina, Czaja, Steele, Wogsland, Lee, Flannery, Baker, Cloutis, Cardarelli, Scheller, Berger, McCubbin, Razzell Hollis, Hickman-Lewis, Steadman, Uckert, DeFlores, Kah, Beegle, Fries, Minitti, Haney, Conrad, Morris, Bhartia, Roppel, Siljeström, Asher, Bykov, Sharma, Shkolyar, Fornaro and Abbey2023; Sharma et al., Reference Sharma, Roppel, Murphy, Beegle, Bhartia, Steele, Razzell Hollis, Siljeström, McCubbin, Asher, Abbey, Allwod, Berger, Bleefeld, Burton, Bykov, Cardarelli, Conrad, Corpolongo, Czaja, DeFlores, Edgett, Farley, Fornaro, Fox, Fries, Harker, Hickman-Lewis, Huggett, Imbeah, Jakubek, Kah, Lee, Liu, Magee, Minitti, Moore, Pascuzzo, Rodriguez, Scheller, Shkolyar, Stack, Steadman, Tuite, Uckert, Werynski, Wiens, Williams, Winchell, Wu and Yanchilina2023), among other instruments (Udry et al., Reference Udry, Ostwald, Sautter, Cousin, Beyssac, Forni, Dromart, Benzerara, Nachon, Horgan, Mandon, Clavé, Dehouck, Gibbons, Alwmark, Ravanis, Wiens, Legett, Anderson, Pilleri, Mangold, Schmidt, Liu, Núñez, Castro, Madariaga, Kizovski, Beck, Bernard, Bosak, Brown, Clegg, Cloutis, Cohen, Connell, Crumpler, Debaille, Flannery, Fouchet, Gabriel, Gasnault, Herd, Johnson, Anrique, Maurice, McCubbin, McLennan, Ollila, Pinet, Quantin-Nataf, Royer, Sharma, Simon, Steele, Tosca and Treiman2022; Beyssac et al., Reference Beyssac, Forni, Cousin, Udry, Kah, Mandon, Clavé, Liu, Poulet, Quantin Nataf, Gasnault, Johnson, Benzerara, Beck, Dehouck, Mangold, Alvarez Llamas, Anderson, Arana, Barnes, Bernard, Bosak, Brown, Castro, Chide, Clegg, Cloutis, Fouchet, Gabriel, Gupta, Lacombe, Lasue, Le Mouelic, Lopez-Reyes, Madariaga, McCubbin, McLennan, Manrique, Meslin, Montmessin, Núñez, Ollila, Ostwald, Pilleri, Pinet, Royer, Sharma, Schröder, Simon, Toplis, Veneranda, Willis, Maurice and Wiens2023; Simon et al., Reference Simon, Hickman-Lewis, Cohen, Mayhew, Shuster, Debaille, Hausrath, Weiss, Bosak, Zorzano, Amundsen, Beegle, Bell, Benison, Berger, Beyssac, Brown, Calef, Casademont, Clark, Clavé, Crumpler, Czaja, Fairén, Farley, Flannery, Fornaro, Forni, Gómez, Goreva, Gorin, Hand, Hamran, Henneke, Herd, Horgan, Johnson, Joseph, Kronyak, Madariaga, Maki, Mandon, McCubbin, McLennan, Moeller, Newman, Núñez, Pascuzzo, Pedersen, Poggiali, Pinet, Quantin-Nataf, Rice, Rice, Royer, Schmidt, Sephton, Sharma, Siljeström, Stack, Steele, Sun, Udry, VanBommel, Wadhwa, Wiens, Williams and Williford2023). In addition to the structures of biomolecules such as nucleotides and amino acids (Sapers et al., Reference Sapers, Razzell Hollis, Bhartia, Beegle, Orphan and Amend2019), DUV fluorescence/Raman spectroscopy is particularly sensitive to aromatic organic compounds (Abbey et al., Reference Abbey, Bhartia, Beegle, DeFlores, Paez, Sijapati, Sijapati, Williford, Tuite, Hug and Reid2017 and references therein), and its use on Mars has suggested the presence of organic matter on Mars (Scheller et al., Reference Scheller, Razzell Hollis, Cardarelli, Steele, Beegle, Bhartia, Conrad, Uckert, Sharma, Ehlmann, Abbey, Asher, Benison, Berger, Beyssac, Bleefeld, Bosak, Brown, Burton, Bykov, Cloutis, Fairén, DeFlores, Farley, Fey, Fornaro, Cox, Fries, Hickman-Lewis, Hug, Huggett, Imbeah, Jakubek, Kah, Kelemen, Kennedy, Kizovski, Lee, Liu, Mandon, McCubbin, Moore, Nixon, Nuñez, Rodriguez Sanchez-Vahamonde, Roppel, Schulte, Sephton, Sharma, Siljestrom, Shkolyar, Shuster, Simon, Smith, Stack, Steadman, Weiss, Werynski, Williams, Wiens, Williford, Winchell, Wogsland, Yanchilina, Yingling and Zorzano2022; Sharma et al., Reference Sharma, Roppel, Murphy, Beegle, Bhartia, Steele, Razzell Hollis, Siljeström, McCubbin, Asher, Abbey, Allwod, Berger, Bleefeld, Burton, Bykov, Cardarelli, Conrad, Corpolongo, Czaja, DeFlores, Edgett, Farley, Fornaro, Fox, Fries, Harker, Hickman-Lewis, Huggett, Imbeah, Jakubek, Kah, Lee, Liu, Magee, Minitti, Moore, Pascuzzo, Rodriguez, Scheller, Shkolyar, Stack, Steadman, Tuite, Uckert, Werynski, Wiens, Williams, Winchell, Wu and Yanchilina2023). With respect to minerals, some mineral classes (e.g., sulphate vs carbonate and pyroxene vs olivine) are identified by DUV fluorescence/Raman spectroscopy based on the number of major peaks and their general positions in Raman spectra (Hollis et al., Reference Hollis, Abbey, Beegle, Bhartia, Ehlmann, Miura, Monacelli, Moore, Nordman and Scheller2021; Corpolongo et al., Reference Corpolongo, Jakubek, Burton, Brown, Yanchilina, Czaja, Steele, Wogsland, Lee, Flannery, Baker, Cloutis, Cardarelli, Scheller, Berger, McCubbin, Razzell Hollis, Hickman-Lewis, Steadman, Uckert, DeFlores, Kah, Beegle, Fries, Minitti, Haney, Conrad, Morris, Bhartia, Roppel, Siljeström, Asher, Bykov, Sharma, Shkolyar, Fornaro and Abbey2023). However, measurable Raman signals are not obtained from a number of silicate minerals including smectites and iron-rich minerals due to significant UV absorption.
Depending on translucency, FT-IR spectroscopy also listed by SSAF and MSPG2 requires a sample thickness ranging from 10 to 30 μm under transmission mode, which often requires destructive sample preparation. FT-IR microscopy has been applied to detect biological materials from basaltic rock core samples obtained by scientific ocean drilling (Preston et al., Reference Preston, Izawa and Banerjee2011; Türke et al., Reference Türke, Ménez and Bach2018). In these studies, the 30 μm-thick sections of the rock core samples were prepared (Preston et al., Reference Preston, Izawa and Banerjee2011; Türke et al., Reference Türke, Ménez and Bach2018). Destructive sample preparation required for the transmission mode of FT-IR microscopy is not necessary for the ATR mode. As previously demonstrated (Tanykova et al., Reference Tanykova, Petrova, Kostina, Kozlova, Leushina and Spasennykh2021), the absorption intensities of FT-IR spectra by the ATR mode are substantially lower than those by the transmission mode. Although this study attempted to obtain FT-IR spectra via the ATR mode, no FT-IR spectra diagnostic to microbial cells or smectite were obtained.
The in-situ capability of O-PTIR spectroscopy to sensitively identify smectites has some advantage over those of DUV fluorescence/Raman spectroscopy and FT-IR microscopy, given that Fe/Mg smectites, one of the most widely reported hydrated minerals on Mars including the Jezero crater, are known for preservation of organic biosignatures (Singh et al., Reference Singh, Sinha, Singh, Roy and Mukherjee2022). In particular, the high spatial resolution and negligible interferences for the detection of organics with high structural complexity such as peptides and smectites are crucial to obtain the co-occurrence pattern, as well as the abiotic background associated with smectites.
After spectroscopic analyses, the SSAF test sequence suggests destructive analyses using organic extracts (Kminek et al., Reference Kminek, Benardini, Brenker, Brooks, Burton, Dhaniyala, Dworkin, Fortman, Glamoclija, Grady, Graham, Haruyama, Kieft, Koopmans, McCubbin, Meyer, Mustin, Onstott, Pearce, Pratt, Sephton, Siljeström, Sugahara, Suzuki, Suzuki, Zuilen and Viso2022a). Mass spectrometers and next-generation DNA sequencers are proposed to detect biomolecules expected in terrestrial life. Martian life could use biomolecules other than nucleic acids, proteins and lipids, on which the methodology of terrestrial life detection is based. Thus, even if no biomolecules known from terrestrial life are detected, agnostic life detection targeting non-terrestrial life will be necessary. If non-terrestrial life occurs in Mars return samples, O-PTIR spectroscopy can detect some characteristics of Martian life such as the enrichment of organic molecules with sufficient structural complexity unexplained by known abiotic processes. In addition, the space and cost required for O-PTIR spectroscopy are comparable to those required for DUV fluorescence/Raman spectroscopy.
Acknowledgments
We are grateful to Takahiro Tajima and Yasushi Suzuki from Shimadzu Corporation and Hanae Kobayashi from Nihon Thermal Consulting Co., Ltd. for their technical assistance about FT-IR microscopy and O-PTIR spectroscopy, respectively. Y. S. was supported by the Astrobiology Center Program of National Institutes of Natural Sciences (NINS) (AB0502). M. A. S. was supported by UK Space Agency grants ST/V002732/1 and ST/V006134/1.
Competing interests
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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