Spectral fingerprinting of cellular lipid droplets using stimulated Raman scattering microscopy and chemometric analysis.

Hyperspectral stimulated Raman scattering (SRS) microscopy is a powerful method for direct visualisation and compositional analysis of cellular lipid droplets. Here we report the application of spectral phasor analysis as a convenient method for the segmentation of lipid droplets using the hyperspectral SRS spectrum in the high wavenumber and fingerprint region of the spectrum. Spectral phasor analysis was shown to discriminate six fatty acids based on vibrational spectroscopic features in solution. The methodology was then applied to studying fatty acid metabolism and storage in a mammalian cancer cell model and during drug-induced steatosis in a hepatocellular carcinoma cell model. The accumulation of fatty acids into cellular lipid droplets was shown to vary as a function of the degree of unsaturation, whilst in a model of drug-induced steatosis, the detection of increased saturated fatty acid esters was observed. Taking advantage of the fingerprint and high wavenumber regions of the SRS spectrum has yielded a greater insight into lipid droplet composition in a cellular context. This approach will find application in the label-free profiling of intracellular lipids in complex disease models.

Experimental determination of solvation free energy of protons in non-protic ionic liquids using Raman spectroscopy.

Room temperature imidazolium-based ionic liquids (ILs) often present super-acidity, which can be characterized by the free energy of solvation of protons in ILs, ΔsolvG°(H+)IL. It can be derived from the consensus value of the free energy of solvation of protons in water if the free energy of transfer of protons from water to the ILs, ΔtG°(H+), is determined. However, the experimental determination of the free energy of transfer of protons relies on extra-thermodynamic hypotheses, as protons cannot be transferred from one solvent to another without a counterion. Here we propose to measure the Hammett acidity functions, which relies on the protonation equilibrium of specific pH-reporters, for the first time by Raman spectroscopy directly in acidic solution of 2,6-dichloro-4-nitroaniline in three 1-alkyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ILs. We demonstrated that the ΔtG°(H+) obtained by Raman spectroscopy and UV-visible spectroscopy were identical in the same 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide. Also, when the butyl substituent is replaced by a longer alkyl chain such as an octyl chain, the acidity in the IL is lowered. The free energies of solvation are calculated in four ILs from Raman spectroscopy data recorded directly in the acidic solutions. These values confirmed that the protons are less solvated in ILs than in water, hence their acidity. Raman spectroscopy also enables determination of the solvation number of the proton in imidazolium-based bis(trifluoromethylsulfonyl)imide ILs. The benefits of implementing Raman spectroscopy to determine the Hammett acidity function in ILs using a non-colored pH-reporter and in colored media are also discussed.

Surface-assisted laser desorption/ionization mass spectrometry imaging: A review.

In the last decades, surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) has attracted increasing interest due to its unique capabilities, achievable through the nanostructured substrates used to promote the analyte desorption/ionization. While the most widely recognized asset of SALDI-MS is the untargeted analysis of small molecules, this technique also offers the possibility of targeted approaches. In particular, the implementation of SALDI-MS imaging (SALDI-MSI), which is the focus of this review, opens up new opportunities. After a brief discussion of the nomenclature and the fundamental mechanisms associated with this technique, which are still highly controversial, the analytical strategies to perform SALDI-MSI are extensively discussed. Emphasis is placed on the sample preparation but also on the selection of the nanosubstrate (in terms of chemical composition and morphology) as well as its functionalization possibilities for the selective analysis of specific compounds in targeted approaches. Subsequently, some selected applications of SALDI-MSI in various fields (i.e., biomedical, biological, environmental, and forensic) are presented. The strengths and the remaining limitations of SALDI-MSI are finally summarized in the conclusion and some perspectives of this technique, which has a bright future, are proposed in this section.

SERS nanotags for folate receptor α detection at the single cell level: discrimination of overexpressing cells and potential for live cell applications.

Folate receptor α (FRα) is a high affinity folate membrane receptor that is overexpressed in a wide variety of cancers. Detecting the overexpression of this receptor is important for cancer cells identification and to potentially guide the choice of treatment since several FRα-targeted drugs are currently in clinical trials. In this work, we built SERS nanotags based on core@shell Au@Ag nanoparticles labelled with resonant Raman-reporter and functionalised with a thiolated PEG linker bearing folic acid at the chain end. Using SERS mapping on single cells, we showed that the nanotags (FR-nanotags) could specifically target FRα on overexpressing HeLa cells and could measure the gradual blocking of FRα by free folic acid introduced in the media along the nanotags. With a control nanotag, we showed that the SERS response was 10-fold higher on HeLa cells when folic acid is present on the PEG linker compared to PEG chains without folic acid. Non-specific binding of the FR-nanotags was demonstrated to be low and mainly caused by the folic acid molecule at the PEG chain end. When comparing cancer cells with different expression levels of FRα, we obtained 4-fold higher SERS response on overexpressing HeLa cells compared to non-overexpressing A549 cells, allowing the discrimination of both cell lines with a high contrast. Owing to the biocompatibility of the developed nanotags, we demonstrated that measurements of FRα on live HeLa cells were also possible and gave similar results to measurements on fixed cells, indicating the versatility of the developed nanotags for detecting FRα under various experimental conditions.

Structure and mineralization of the spearing mantis shrimp (Stomatopoda; Lysiosquillina maculata) body and spike cuticles.

Stomatopoda is a crustacean order including sophisticated predators called spearing and smashing mantis shrimps that are separated from the well-studied Eumalacotraca since the Devonian. The spearing mantis shrimp has developed a spiky dactyl capable of impaling fishes or crustaceans in a fraction of second. In this high velocity hunting technique, the spikes undergo an intense mechanical constraint to which their exoskeleton (or cuticle) has to be adapted. To better understand the spike cuticle internal architecture and composition, electron microscopy, X-ray microanalysis and Raman spectroscopy were used on the spikes of 7 individuals (collected in French Polynesia and Indonesia), but also on parts of the body cuticle that have less mechanical stress to bear. In the body cuticle, several specificities linked to the group were found, allowing to determine the basic structure from which the spike cuticle has evolved. Results also highlighted that the body cuticle of mantis shrimps could be a model close to the ancestral arthropod cuticle by the aspect of its biological layers (epi- and procuticle including exo- and endocuticle) as well as by the Ca-carbonate/phosphate mineral content of these layers. In contrast, the spike cuticle exhibits a deeply modified organization in four functional regions overprinted on the biological layers. Each of them has specific fibre arrangement or mineral content (fluorapatite, ACP or phosphate-rich Ca-carbonate) and is thought to assume specific mechanical roles, conferring appropriate properties on the entire spike. These results agree with an evolution of smashing mantis shrimps from primitive stabbing/spearing shrimps, and thus also allowed a better understanding of the structural modifications described in previous studies on the dactyl club of smashing mantis shrimps.

Dual-polarity SALDI FT-ICR MS imaging and Kendrick mass defect data filtering for lipid analysis.

Lipids are biomolecules of crucial importance involved in critical biological functions. Yet, lipid content determination using mass spectrometry is still challenging due to their rich structural diversity. Preferential ionisation of the different lipid species in the positive or negative polarity is common, especially when using soft ionisation mass spectrometry techniques. Here, we demonstrate the potency of a dual-polarity approach using surface-assisted laser desorption/ionisation coupled to Fourier transform-ion cyclotron resonance (SALDI FT-ICR) mass spectrometry imaging (MSI) combined with Kendrick mass defect data filtering to (i) identify the lipids detected in both polarities from the same tissue section and (ii) show the complementarity of the dual-polarity data, both regarding the lipid coverage and the spatial distributions of the various lipids. For this purpose, we imaged the same mouse brain section in the positive and negative ionisation modes, on alternate pixels, in a SALDI FT-ICR MS imaging approach using gold nanoparticles (AuNPs) as dual-polarity nanosubstrates. Our study demonstrates, for the first time, the feasibility of (i) a dual-polarity SALDI-MSI approach on the same tissue section, (ii) using AuNPs as nanosubstrates combined with a FT-ICR mass analyser and (iii) the Kendrick mass defect data filtering applied to SALDI-MSI data. In particular, we show the complementarity in the lipids detected both in a given ionisation mode and in the two different ionisation modes. Graphical abstract.

Spatially resolved determination of the abundance of the HER2 marker in microscopic breast tumors using targeted SERS imaging.

Highly selective nanoprobes have been developed based on SERS-active Au@Ag nanoparticles protected by a PEG coating and functionalized with monoclonal antibodies against human epidermal growth factor receptor 2 (HER2). The PEG coating allows to drastically reduce unspecific interactions during incubation on tissues, while the monoclonal antibodies allow a highly specific targeting of HER2. Using the designed SERS nanoprobes combined with a spectral imaging and data weighting approach, we demonstrate the proportionality between the SERS signal and the amount of HER2 antigen on the cell membranes as measured by digital image analysis of IHC staining in microscopic breast tumors (linear fit R2 = 0.87). We also show that the level of expression of HER2 measured by SERS is significantly different between several microscopic tumor parts of the same tissue slide. Therefore, SERS is proving to be a suitable technique for the localized quantitative measurement of specific markers in breast cancerous tissues. Owing to its high multiplexing capabilities, SERS could be a future tool of choice for characterizing the molecular heterogeneity of tumors at the microscopic scale.

Microstructural and compositional variation in pacu and piranha teeth related to diet specialization (Teleostei: Serrasalmidae).

In any vertebrate group, tooth shape is known to fit with a biological function related to diet. However, little is known about the relationships between diet and tooth microstructure and composition in teleost fishes. In this work, we describe the external morphology, internal microstructure and elemental composition of the oral teeth of three representative species of the family Serrasalmidae having different feeding habits (herbivorous vs. omnivorous vs. carnivorous). We used backscattered-electron imaging and low vacuum environmental scanning electron microscope to compare the organization and mineralization of tooth layers as well as energy dispersive X-ray microanalysis and Raman microspectrometry to investigate the elemental composition, Ca/P ratio and mineralogy of the most superficial layers. Oral teeth of each serrasalmid species have the same internal organization based on five distinctive layers (i.e. pulp, dentine, inner enameloid, outer enameloid and cuticle) but the general tooth morphology is different according to diet. Microstructural and compositional variation of the cuticle and iron-enrichment of superficial layers were highlighted between herbivorous and carnivorous species. Iron is more concentrated in teeth of the herbivorous species where it is associated with a thicker cuticle explaining the more intense red-pigmentation of the cutting edges of oral teeth. The iron-enrichment is interpreted as a substitution of Ca by Fe in the hydroxyapatite. These traits are discussed in the light of the evolutionary history of the family. Further considerations and hypotheses about the formation and origin of the mineralized tooth layers and especially the iron-rich superficial layers in teleost fishes are suggested.

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a "standardization" process of SERS protocols, not proposed by a single laboratory but by a larger community.

Multiplex micro-SERS imaging of cancer-related markers in cells and tissues using poly(allylamine)-coated Au@Ag nanoprobes.

Surface-enhanced Raman scattering (SERS) nanoprobes based on Au@Ag core@shell nanoparticles coated with poly(allylamine) were functionalized with small targeting molecules to evaluate simultaneously the level of expression of two cancer-related markers, both in cells and in tissues. The Au@Ag nanoparticles provide a high SERS signal enhancement in the visible range when combined with resonant Raman-active molecules. The poly(allylamine) coating plays a dual key role in (i) protecting the metal surface against the complex biological medium, leading to a stable signal of the Raman-active molecules, and (ii) enabling specific biofunctionalization through its amine functions. Using small targeting molecules linked to the polymer coating, two different nanoprobes (duplex approach) were designed. Each was able to specifically target a particular cancer-related marker: folate receptors (FRs) and sialic acid (SA). We demonstrate that the level of expression of these targeted markers can be evaluated following the SERS signal of the probes incubated on cells or tissues. The potential overexpression of folate receptors and of sialic acid was evaluated and measured in breast and ovarian cancerous tissue sections. In addition, FR and/or SA overexpression in the tumor region can be visualized with high contrast with respect to the healthy region and with high spatial accuracy consistent with histology by SERS imaging of the nanoprobe signal. Owing to the unique spectral signature of the designed nanoprobes, this approach offers an efficient tool for the spatially resolved, in situ measurement of the expression level of several cancer-related markers in tumors at the same time.Graphical abstract.

Understanding chemical interaction between phosphonate-derivative molecules and a silver surface cluster in SERS: a combined experimental and computational approach.

The interaction between phosphonate functions and a silver surface cluster is investigated using Surface-Enhanced Raman Spectroscopy (SERS). Changing the functional group (methylphosphonic acid based molecule) by studying the effect of protonation, methylation and substitution of the side chain with amine and carboxylate functions enabled us to modulate the chemical interactions between the different functions and the metal cluster. We find that the adsorption energy of the methylphosphonic acid decreases with the protonation, the methylation processes and the substitution of the side chain. In all cases, only the deprotonated phosphonate forms are SERS active. To understand how the molecules interact with the nanoparticle, the electronic structure, adsorption energies and Raman spectra were computed for molecules adsorbed on a 20 atom silver cluster representing a nanoparticle surface. The qualitative agreement between computed static Raman spectra and experimental SERS spectra makes it possible to determine stable geometries of the analyte-silver cluster complexes and to characterize the adsorption modes. The findings presented here provide a framework for designing analytical developments based on SERS for simultaneous detection of phosphonated molecules, including pesticides such as glyphosate, creating practical opportunities in key areas such as environmental and water resource in situ monitoring.

Determination of the Al2O3 content in NaF-AlF3-CaF2-Al2O3 melts at 950 °C by Raman spectroscopy.

The in situ control of the chemical composition of industrial aluminum smelter is a challenge mainly for physicochemical reasons: high temperature, high surrounding electromagnetic field, and the highly corrosive molten salt electrolyte to deal with. In previous works, we proposed that Raman spectroscopy is a method of choice that could be adapted to real smelters. The laboratory study presented here relies on reproducible Raman spectra recorded on molten mixtures whose compositions are identical to those used during the production of aluminum. A normalization procedure for the Raman spectra is proposed based on the equilibria taking place in the bath. In addition, we discuss two quantitative models to determine the alumina content from the Raman spectra of the molten NaF-AlF3-CaF2-Al2O3 electrolytes. Univariate and multivariate approaches are applied to determine both the COx (alumina content) and the CR (NaF/AlF3 molar ratio) by Raman spectroscopy without referring to an additional internal reference of intensity. The procedure was successfully tested and validated on industrial samples.

Designing SERS nanotags for profiling overexpressed surface markers on single cancer cells: A review.

This review focuses on the chemical design and the use of Surface-Enhanced Raman Scattering (SERS)-active nanotags for measuring surface markers that can be overexpressed at the surface of single cancer cells. Indeed, providing analytical tools with true single-cell measurements capabilities is capital, especially since cancer research is increasingly leaning toward single-cell analysis, either to guide treatment decisions or to understand complex tumor behaviour including the single-cell heterogeneity and the appearance of treatment resistance. Over the past two decades, SERS nanotags have triggered significant interest in the scientific community owing their advantages over fluorescent tags, mainly because SERS nanotags resist photobleaching and exhibit sharper signal bands, which reduces possible spectral overlap and enables the discrimination between the SERS signals and the autofluorescence background from the sample itself. The extensive efforts invested in harnessing SERS nanotags for biomedical purposes, particularly in cancer research, highlight their potential as the next generation of optical labels for single-cell studies. The review unfolds in two main parts. The first part focuses on the structure of SERS nanotags, detailing their chemical composition and the role of each building block of the tags. The second part explores applications in measuring overexpressed surface markers on single-cells. The latter encompasses studies using single nanotags, multiplexed measurements, quantitative information extraction, monitoring treatment responses, and integrating phenotype measurements with SERS nanotags on single cells isolated from complex biological matrices. This comprehensive review anticipates SERS nanotags to persist as a pivotal technology in advancing single-cell analytical methods, particularly in the context of cancer research and personalized medicine.

Direct determination of the NaF/AlF3 molar ratio by Raman spectroscopy in NaF-AlF3-CaF2 melts at 1000 °C.

For the last 40 years, Raman spectroscopy has been very useful in investigating the structure of corrosive molten salts, such as the cryolite-based melts widely used as electrolyte in the Hall-Heroult process. Even if this process remains the most economically efficient for metallic aluminum electro-production, it suffers from a high energy loss, which is dependent on the melt composition. Therefore, controlling the chemical composition of the electrolyte is essential. The present paper proposes to apply Raman spectroscopy for the direct determination of the NaF-AlF3 molar ratio in NaF-AlF3-CaF2-based melts. Despite the experimental difficulties, a calibration curve based on equilibria taking place in the melt has been developed and the procedure has been successfully compared to industrial samples of known compositions. The possible exportation of the laboratory scale procedure to an industrial environment application for the control of the Hall-Heroult process is finally discussed.

Evaluation of meso- and microplastic ingestion by the northern fulmar through a non-lethal sampling method.

An increasing number of organisms from the polar regions are reported contaminated by plastic. Rarely a non-killing sampling method is used. In this study we wanted to assess plastic levels using stomach flushing and evaluate the method suitability for further research and monitoring. The stomach of 22 fulmars from Bjørnøya, Svalbard, were flushed with water in the field. On return to the laboratory, the regurgitated content was digested using potassium hydroxide. The extracted plastics were visually characterised and analysed with spectroscopy. Only three birds had plastics in their stomach, totaling 36 particles, most of them microplastics (< 5 mm). The plastic burdens are much lower than previously reported in Svalbard. The stomach flushing is assumed not to allow the collection of the gizzard content. This is a major limitation as most of the plastics accumulate in the fulmar's gizzard. However, the method is still useful for studies investigating plastic ingestion dynamics, allowing to sample the same individuals over time.

Online monitoring of heterogeneous polymerizations in supercritical carbon dioxide by Raman spectroscopy.

A novel setup is described to monitor the kinetics of homogeneous and heterogeneous reactions online using Raman spectroscopy under high pressure. The arrangement is based on a high-pressure reactor equipped with a sapphire window in combination with a mobile probe that allows the collection of the back-scattered Raman signal at 360°.

Macroporous Mannitol Granules Produced by Spray Drying and Sacrificial Templating.

In pharmaceutical applications, the porous particles of organic compounds can improve the efficiency of drug delivery, for example into the pulmonary system. We report on the successful preparation of macroporous spherical granules of mannitol using a spray-drying process using polystyrene (PS) beads of ~340 nm diameter as a sacrificial templating agent. An FDA-approved solvent (ethyl acetate) was used to dissolve the PS beads. A combination of infrared spectroscopy and thermogravimetry analysis proved the efficiency of the etching process, provided that enough PS beads were exposed at the granule surface and formed an interconnected network. Using a lab-scale spray dryer and a constant concentration of PS beads, we observed similar granule sizes (~1-3 microns) and different porosity distributions for the mannitol/PS mass ratio ranging from 10:1 to 1:2. When transferred to a pilot-scale spray dryer, the 1:1 mannitol/PS composition resulted in different distributions of granule size and porosity depending on the atomization configuration (two-fluid or rotary nozzle). In all cases, the presence of PS beads in the spray-drying feedstock was found to favor the formation of the α mannitol polymorph and to lead to a small decrease in the mannitol decomposition temperature when heating in an inert atmosphere.

Fe-Rich Fossil Vents as Mars Analog Samples: Identification of Extinct Chimneys in Miocene Marine Sediments Using Raman Spectroscopy, X-Ray Diffraction, and Scanning Electron Microscopy-Energy Dispersive X-Ray Spectroscopy.

On Earth, the circulation of Fe-rich fluids in hydrothermal environments leads to characteristic iron mineral deposits, reflecting the pH and redox chemical conditions of the hydrothermal system, and is often associated with chemotroph microorganisms capable of deriving energy from chemical gradients. On Mars, iron-rich hydrothermal sites are considered to be potentially important astrobiological targets for searching evidence of life during exploration missions, such as the Mars 2020 and the ExoMars 2022 missions. In this study, an extinct hydrothermal chimney from the Jaroso hydrothermal system (SE Spain), considered an interesting geodynamic and mineralogical terrestrial analog for Mars, was analyzed using Raman spectroscopy, X-ray diffraction, and scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. The sample consists of a fossil vent in a Miocene shallow-marine sedimentary deposit composed of a marl substrate, an iron-rich chimney pipe, and a central space filled with backfilling deposits and vent condensates. The iron crust is particularly striking due to the combined presence of molecular and morphological indications of a microbial colonization, including mineral microstructures (e.g., stalks, filaments), iron oxyhydroxide phases (altered goethite, ferrihydrite), and organic signatures (carotenoids, organopolymers). The clear identification of pigments by resonance Raman spectroscopy and the preservation of organics in association with iron oxyhydroxides by Raman microimaging demonstrate that the iron crust was indeed colonized by microbial communities. These analyses confirm that Raman spectroscopy is a powerful tool for documenting the habitability of such historical hydrothermal environments. Finally, based on the results obtained, we propose that the ancient iron-rich hydrothermal pipes should be recognized as singular terrestrial Mars analog specimens to support the preparatory work for robotic in situ exploration missions to Mars, as well as during the subsequent interpretation of data returned by those missions.

Development and validation of an integrated microfluidic device with an in-line Surface Enhanced Raman Spectroscopy (SERS) detection of glyphosate in drinking water.

Glyphosate, also known as N-(phosphonomethyl)glycine, is one of the most widely used herbicides in the world. However, the controversy surrounding the toxicity of glyphosate and its main breakdown product, aminomethylphosphonic acid (AMPA), remains a serious public concern. Therefore, there is a clear need to develop a rapid, sensitive and automated alternative method for the quantification of glyphosate and AMPA. In this context, surface enhanced Raman spectroscopy (SERS) coupled with a microfluidic system for the determination of glyphosate in tap water was developed, optimized and validated. The design of the microfluidic configuration for this application was built constructed to integrate the synthesis of the SERS substrate through to the detection of the analyte. To optimize the microfluidic setup, a design of experiments approach was used to maximize the SERS signal of glyphosate. Subsequently, an approach based on the European guideline document SANTE/11312/2021 was used to validate the method in the range of 78-480 µg/L using the normalized band intensities. The limit of detection and quantification obtained for glyphosate were 40 and 78 µg/L, respectively. Recoveries were in the range 76-117%, while repeatability and intra-day reproducibility were ≤17%. Finally, the method was also tested for the determination of AMPA in tap water matrix and for the simultaneous detection of AMPA and glyphosate.

Characterization of the Halochromic Gloeocapsin Pigment, a Cyanobacterial Biosignature for Paleobiology and Astrobiology.

Ultraviolet (UV)-screening compounds represent a substantial asset for the survival of cyanobacteria in extreme environments exposed to high doses of UV radiations on modern and early Earth. Among these molecules, the halochromic pigment gloeocapsin remains poorly characterized and studied. In this study, we identified a gloeocapsin-producing cultivable cyanobacteria: the strain Phormidesmis nigrescens ULC007. We succeeded to extract, to partially purify, and to compare the dark blue pigment from both the ULC007 culture and an environmental Gloeocapsa alpina dominated sample. FT-IR and Raman spectra of G. alpina and P. nigrescens ULC007 pigment extracts strongly suggested a common backbone structure. The high-pressure liquid chromatography-UV-MS/MS analysis of the ULC007 pigment extract allowed to narrow down the molecular formula of gloeocapsin to potentially five candidates within three classes of halochromic molecules: anthraquinone derivatives, coumarin derivatives, and flavonoids. With the discovery of gloeocapsin in P. nigrescens, the production of this pigment is now established for three lineages of cyanobacteria (including G. alpina, P. nigrescens, and Solentia paulocellulare) that belong to three distinct orders (Chroococcales, Pleurocapsales, Synechoccocales), inhabiting very diverse environments. This suggests that gloeocapsin production was a trait of their common ancestor or was acquired by lateral gene transfer. This work represents an important step toward the elucidation of the structure of this enigmatic pigment and its biosynthesis, and it potentially provides a new biosignature for ancient cyanobacteria. It also gives a glimpse on the evolution of UV protection strategies, which are relevant for early phototrophic life on Earth and possibly beyond.