Raman imaging allows one to obtain spatially resolved chemical information in a nondestructive manner. Herein, we present analytical aspects of effective in situ and in vivo Raman imaging of algae and cyanobacteria from within their native rock habitats. Specifically, gypsum and halite inhabited by endolithic communities from the hyperarid Atacama Desert were analyzed. Raman imaging of these phototrophic colonization reveals a pigment composition within the aggregates that helps in understanding some of their adaptation strategies to survive in this harsh polyextreme environment. The study is focused on methodical aspects of Raman imaging acquisition and subsequent data processing. Point imaging is compared with line imaging in terms of their image quality, spatial resolution, spectral signal-to-noise ratio, time requirements, and risk of laser-induced sample alteration. The roles of excitation wavelength, exposure time, and step size of the imaging grid on successful Raman imaging results are also discussed. Graphical abstract.

Departamento Biología Vegetal Ecología y Ciencias de la Tierra and IACYS Universidad de Extremadura 10600 Plasencia Spain

Global Change Research Institute of the Czech Academy of Sciences Bělidla 986 4a 603 00 Brno Czech Republic

Museo Nacional de Ciencias Naturales CSIC c Serrano 115 dpdo 28006 Madrid Spain

Zobrazit více v PubMed

Delhaye M, Dhamelincourt P. Raman microprobe and microscope with laser excitation. J Raman Spectrosc. 1975;3:33–43. DOI

Markwort L, Kip B, Dasilva E, Roussel B. Raman imaging of heterogeneous polymers – a comparison of global versus point illumination. Appl Spectrosc. 1995;49:1411–30. DOI

Schlücker S, Schaeberle MD, Huffmann SW, Levin IW. Raman microspectroscopy: a comparison of point, line, and wide-field imaging methodologies. Anal Chem. 2003;75:4312–8. DOI

Puppels GJ, Grond M, Greve J. Direct imaging Raman microscope based on tunable wavelength excitation and narrow-band emission detection. Appl Spectrosc. 1993;47:1256–67. DOI

Baranski R, Baranska M, Schulz H. Changes in carotenoid content and distribution in living plant tissue can be observed and mapped in situ using NIR-FT-Raman spectroscopy. Planta. 2005;222:448–57. DOI

Schulz H, Baranska M, Baranski R. Potential of NIR-FT-Raman spectroscopy in natural carotenoid analysis. Biopolymers. 2005;7:212–21. DOI

Agarwall UP. Raman imaging to investigate ultrastructure and composition of plant cell walls: distribution of lignin and cellulose in black spruce wood (Picea mariana). Planta. 2006;224:1141–53. DOI

Gierlinger N, Schwaninger M. Chemical imaging of poplar wood cell walls by confocal Raman microscopy. Plant Physiol. 2006;140:1246–54. DOI

Häninen T, Kontturi E, Vuorinen T. Distribution of lignin and its coniferyl alcohol and coniferyl aldehyde groups in Picea abies and Pinus sylvestris as observed by Raman imaging. Phytochemistry. 2011;72:1889–95. DOI

Gierlinger N, Keplinger T, Harrington M. Imaging of plant cell walls by confocal Raman microscopy. Nat Protoc. 2012;7:1694–708. DOI

Gierlinger N, Keplinger T, Harrington M, Schwanninger M. Raman imaging of lignocellulosic feedstock. In: van de Ven T, Kadla J, editors. Cellulose biomass conversion 3. Rijeka: INTECH; 2013. p. 159–92.

Ji Z, Ma JF, Zhang ZH, Xu F, Sun RC. Distribution of lignin and cellulose in compression wood tracheids of Pinus yunnanensis determined by fluorescence microscopy and confocal Raman microscopy. Ind Crop Prod. 2013;47:212–7. DOI

Gowen AA, Feng Y, Gaston E, Valdramidis V. Recent applications of hyperspectral imaging in microbiology. Talanta. 2015;137:43–54. DOI

Wang A, Korotev RL, Jolliff BL, Ling Z. Raman imaging of extraterrestrial materials. Planet Space Sci. 2015;112:23–34. DOI

Marshall CP, Olcott MA. (2013). Raman hyperspectral imaging of microfossils: potential pitfalls. Astrobiology. 2013;13:920–31. DOI

Emry JR, Olcott Marshall A, Marchall CP. Evaluating the effects of autofluorescence during Raman hyperspectral imaging. Geostand Geoanal Res. 2015;40:29–47. DOI

Hofmann A, Bolhar R, Orberger F. Cherts of the Barberton greenstone belt, South Africa: petrology and trace-element geochemistry of 3.5 to 3.3 Ga old silicified volcanoclastic sediments. S Afr J Geol. 2013;116:297–322. DOI

Schopf JW, Kudryavtsev AB, Walter MR, Van Kranendonk MJ, Williford KH, Kozdon R, et al. Sulfur-cycling fossil bacteria from the 1.8-Ga Duck Creek Formation provide promising evidence of evolution’s null hypothesis. Proc Natl Acad Sci U S A. 2015;112:2087–92. DOI

Foucher F, Westall F. Raman imaging of metastable opal in carbonaceous microfossils of the 700-800 Ma old Draken formation. Astrobiology. 2013;13:57–67. DOI

Foucher F, Lopez-Reyes G, Bost N, Rull-Perez F, Rüβmann P, Westall F. Effect of grain size distribution on Raman analyses and the consequences for in situ planetary missions. J Raman Spectrosc. 2013;44:916–25. DOI

Westall F, Foucher F, Bost N, Bertrand M, Loizeau D, Vago JL, et al. Biosignatures on Mars: what, where, and how? Implications for search for Martian life. Astrobiology. 2015;15:998–1029. DOI

Vítek P, Ascaso C, Artieda O, Wierzchos J. Raman imaging in geomicrobiology: endolithic phototrophic microorganisms in gypsum from the extreme sun irradiation area in the Atacama Desert. Anal Bioanal Chem. 2016;408:483–92. DOI

Vítek P, Ascaso C, Artieda O, Casero MC, Wierzchos J. Discovery of carotenoid red-shift in endolithic cyanobacteria from the Atacama Desert. Sci Rep. 2017;7:11116. DOI

Rooney JS, Tarling MS, Smith SAF, Gordon KC. Submicron Raman spectroscopy mapping of serpentinite fault rocks. J Raman Spectrosc. 2017;49:279–86. DOI

Mosca S, Artesani A, Gulotta D, Nevin A, Goidanich S, Valentini G, et al. Raman mapping and time-resolved photoluminescence imaging for the analysis of a cross-section from a modern gypsum sculpture. Microchem J. 2018;139:500–5. DOI

Rousaki A, Botteon A, Colombo C, Conti C, Matousek P, Moens L, et al. Development of defocusing micro-SORS mapping: a study of a 19th century porcelain card. Anal Methods. 2017;9:6435–42. DOI

Lauwers D, Brondeel P, Moens L, Vandenabeele P. In situ Raman mapping of art objects. Phil Trans R Soc A. 2016;374:20160039. DOI

Wierzchos J, DiRuggiero J, Vítek P, Artieda O, Souza-Egipsy V, Škaloud P, et al. Adaptation strategies of endolithic chlorophototrophs to survive the hyperarid and extreme solar radiation environment of the Atacama Desert. Front Microbiol. 2015;6:934. DOI

Wierzchos J, Ascaso C, McKay CP. Endolithic cyanobacteria in halite rocks from the hyperarid core of the Atacama Desert. Astrobiology. 2006;6:415–22. DOI

Vítek P, Edwards HGM, Jehlička J, Ascaso C, De los Ríos A, Valea S, et al. Microbial colonization of halite from the hyper-arid Atacama Desert studied by Raman spectroscopy. Phil Trans R Soc A. 2010;368:3205–21. DOI

Vítek P, Jehlička J, Ascaso C, Mašek V, Gómez-Silva B, Olivares H, et al. Distribution of scytonemin in endolithic microbial communities from halite crusts in the hyperarid zone of the Atacama Desert, Chile. FEMS Microbiol Ecol. 2014;90:351–66. PubMed

Vítek P, Jehlička J, Edwards HGM, Hutchinson I, Ascaso C, Wierzchos J. Miniaturized Raman system and the detection of traces of life in halite from the Atacama Desert: some considerations for the search for life signatures on Mars. Astrobiology. 2012;12:1095–9. DOI

Artieda O, Davila A, Wierzchos J, Buhler P, Rodríguez-Ochoa R, Pueyo J, et al. Surface evolution of salt-encrusted playas under extreme and continued dryness. Earth Surf Process Landf. 2015;40:1939–50. DOI

Robinson CK, Wierzchos J, Black C, Crits-Christoph A, Ma B, Ravel J, et al. Microbial diversity and the presence of algae in halite endolithic communities are correlated to atmospheric moisture in the hyper-arid zone of the Atacama Desert. Environ Microbiol. 2015;17:299–315. DOI

Lee E. Imaging modes. In: Zoubir A, editor. Raman imaging, techniques and applications. Springer series in optical sciences 168. Berlin: Springer; 2012. p. 1–37.

Prats-Mateu B, Gierlinger N. Tip in-light on: advantages, challenges, and applications of combining AFM and Raman microscopy on biological samples. Microsc Res Tech. 2017;80:30–40. DOI

Zhang X, Chen S, Ling Z, Zhou X, Ding D-Y, Kim YS, et al. Method for removing spectral contaminants to improve analysis of Raman imaging data. Sci Rep. 2016;7:39819.

Bonnier F, Mehmood A, Knief P, Meade AD, Hornebeck W, Lambkin H, et al. In vitro analysis of immersed human tissues by Raman microspectroscopy. J Raman Spectrosc. 2011;42:888–96. DOI

Nasdala L, Beyssac O, Schopf JW, Bleisteiner B. Application of Raman-based images in the Earth sciences. In: Zoubir A, editor. Raman imaging, techniques and applications. Springer series in optical sciences 168. Berlin: Springer; 2012. p. 145–87. DOI

Foucher F, Guimbretiére G, Bost N, Westall F. Petrographical and mineralogical applications of Raman mapping. In: Raman spectroscopy and applications, Chapter 8, Intech, 2017; pp. 163–180.

Vítek P, Veselá B, Klem K. Spatial and temporal variability of plant responses cascade after PSII inhibition: Raman, chlorophyll fluorescence and infrared thermal imaging. Sensors. 2020;20:1015. DOI

Carotenoids dispersed in gypsum rock as a result of algae adaptation to the extreme conditions of the Atacama Desert

Microbial colonization of gypsum: from the fossil record to the present day

Gypsum endolithic phototrophs under moderate climate (Southern Sicily): their diversity and pigment composition