Modular Spectral Imaging System for Discrimination of Pigments in Cells and Microbial Communities

ABSTRACT Here we describe a spectral imaging system for minimally invasive identification, localization, and relative quantification of pigments in cells and microbial communities. The modularity of the system allows pigment detection on spatial scales ranging from the single-cell level to regions whose areas are several tens of square centimeters. For pigment identification in vivo absorption and/or autofluorescence spectra are used as the analytical signals. Along with the hardware, which is easy to transport and simple to assemble and allows rapid measurement, we describe newly developed software that allows highly sensitive and pigment-specific analyses of the hyperspectral data. We also propose and describe a number of applications of the system for microbial ecology, including identification of pigments in living cells and high-spatial-resolution imaging of pigments and the associated phototrophic groups in complex microbial communities, such as photosynthetic endolithic biofilms, microbial mats, and intertidal sediments. This system provides new possibilities for studying the role of spatial organization of microorganisms in the ecological functioning of complex benthic microbial communities or for noninvasively monitoring changes in the spatial organization and/or composition of a microbial community in response to changing environmental factors.

[1]  R. Guillard,et al.  Culture of Phytoplankton for Feeding Marine Invertebrates , 1975 .

[2]  G. Muyzer,et al.  Structural and functional analysis of a microbial mat ecosystem from a unique permanent hypersaline inland lake: 'La Salada de Chiprana' (NE Spain). , 2003, FEMS microbiology ecology.

[3]  S. Miklavcic,et al.  Revised Kubelka-Munk theory. II. Unified framework for homogeneous and inhomogeneous optical media. , 2004, Journal of The Optical Society of America A-optics Image Science and Vision.

[4]  S. Wright,et al.  Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods , 1997 .

[5]  S. Golubić,et al.  Lipid biomarkers, pigments and cyanobacterial diversity of microbial mats across intertidal flats of the arid coast of the Arabian Gulf (Abu Dhabi, UAE). , 2008, FEMS microbiology ecology.

[6]  Markus Huettel,et al.  Oxygen dynamics in permeable sediments with wave‐driven pore water exchange , 2004 .

[7]  H. Womersley.  Handbook of phycological methods. Culture methods and growth measurements: J.R. Stein (Editor). Cambridge University Press, Cambridge, 1979, x + 448 pp., £7.50 (paperback), ISBN 0-521-29747-8 , 1981 .

[8]  O. Hoegh‐Guldberg,et al.  Fluorescence In Situ Hybridization and Spectral Imaging of Coral-Associated Bacterial Communities , 2006, Applied and Environmental Microbiology.

[9]  N. Dubilier,et al.  Transport and mineralization rates in North Sea sandy intertidal sediments, Sylt‐Rømø Basin, Wadden Sea , 2005 .

[10]  Veronique Carrere,et al.  Spectrometric constraint in analysis of benthic diatom biomass using monospecific cultures , 2003 .

[11]  Pierson,et al.  Phototrophs in high iron microbial mats: microstructure of mats in iron-depositing hot springs. , 2000, FEMS microbiology ecology.

[12]  K. Toda,et al.  Application of a Portable Spectrophotometer to Microbial Mat studies: Temperature Dependence of the Distribution of Cyanobacteria and Photosynthetic Bacteria in Hot Spring Water , 2001 .

[13]  V. Brotas,et al.  Effect of sediment type on microphytobenthos vertical distribution: Modelling the productive biomass and improving ground truth measurements , 2006 .

[14]  A. Maruyama,et al.  Spectral imaging detection and counting of microbial cells in marine sediment. , 2003, Journal of microbiological methods.

[15]  Olaf Pfannkuche,et al.  A marine microbial consortium apparently mediating anaerobic oxidation of methane , 2000, Nature.

[16]  Veronique Carrere,et al.  Comparison of simple techniques for estimating chlorophyll a concentration in the intertidal zone using high spectral-resolution field-spectrometer data , 2004 .

[17]  J. Lawrence,et al.  Microscale Analyses of the Formation and Nature of Microbial Biofilm Communities in River Systems , 2003 .

[18]  B. Jørgensen,et al.  Light penetration and light intensity in sandy marine sediments measured with irradiance and scalar irradiance fiber-optic microprobes , 1994 .

[19]  R. Guillard,et al.  Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. , 1962, Canadian journal of microbiology.

[20]  F. Shiraishi,et al.  Microbial mediation of stromatolite formation in karst‐water creeks , 2008 .

[21]  Anthony W. D. Larkum,et al.  Chlorophyll a Fluorescence A Signature of Photosynthesis. , 2006 .

[22]  K. Timmis,et al.  Three Stages of a Biofilm Community Developing at the Liquid-Liquid Interface between Polychlorinated Biphenyls and Water , 2005, Applied and Environmental Microbiology.

[23]  C. Moldaenke,et al.  "Spectral fingerprinting" for specific algal groups on sediments in situ: a new sensor , 2006 .

[24]  B. Jørgensen,et al.  Photosynthesis and structure of benthic microbial mats: Microelectrode and SEM studies of four cyanobacterial communities1 , 1983 .

[25]  H. Jonkers,et al.  Diversity and Function of Chloroflexus-Like Bacteria in a Hypersaline Microbial Mat: Phylogenetic Characterization and Impact on Aerobic Respiration , 2007, Applied and Environmental Microbiology.

[26]  Kenichi Kobayashi,et al.  In Situ Detection and Identification of Microorganisms at Single Colony Resolution Using Spectral Imaging Technique , 2005, SCIA.

[27]  Rodney M. Forster,et al.  Relationship of intertidal surface sediment chlorophyll concentration to hyperspectral reflectance and chlorophyll fluorescence , 2006 .

[28]  B. Pierson,et al.  Spectral Irradiance and Distribution of Pigments in a Highly Layered Marine Microbial Mat , 1990, Applied and environmental microbiology.

[29]  Warren L. Butler,et al.  AN ANALYSIS OF FOURTH DERIVATIVE SPECTRA , 1970 .

[30]  H. Røy,et al.  Benthic photosynthesis in submerged Wadden Sea intertidal flats , 2007 .

[31]  L. Barillé,et al.  Comparative analysis of field and laboratory spectral reflectances of benthic diatoms with a modified Gaussian model approach , 2007 .

[32]  M. Kühl,et al.  Biogeochemistry of an Iron-Rich Hypersaline Microbial Mat (Camargue, France) , 2003, Microbial Ecology.

[33]  Rodney M. Forster,et al.  Imaging spectroscopy as a tool to study sediment characteristics on a tidal sandbank in the Westerschelde , 2006 .

[34]  S. Passy,et al.  SPECTRAL FINGERPRINTING OF ALGAL COMMUNITIES: A NOVEL APPROACH TO BIOFILM ANALYSIS AND BIOMONITORING 1 , 2005 .

[35]  J. Stein.  Handbook of Phycological methods - Culture methods and Growth measurements , 1973 .

[36]  Serge Andréfouët,et al.  Airborne hyperspectral detection of microbial mat pigmentation in Rangiroa atoll (French Polynesia) , 2003 .

[37]  Richard J. Murphy,et al.  Estimation of surface chlorophyll‐a on an emersed mudflat using field spectrometry: accuracy of ratios and derivative‐based approaches , 2005 .

[38]  M. Kühl,et al.  Bio-optical Characteristics and the Vertical Distribution of Photosynthetic Pigments and Photosynthesis in an Artificial Cyanobacterial Mat , 2000, Microbial Ecology.

[39]  C. S. French,et al.  Fluorescence-Spectrum Curves of Chlorophylls, Pheophytins, Phycoerythrins, Phycocyanins and Hypericin. , 1956, Plant physiology.

[40]  Li Yang,et al.  Revised Kubelka-Munk theory. I. Theory and application. , 2004, Journal of the Optical Society of America. A, Optics, image science, and vision.

[41]  D. de Beer,et al.  High spatial resolution measurement of oxygen consumption rates in permeable sediments , 2005 .

[42]  Gerhard A. Holst,et al.  Luminescence lifetime imaging with transparent oxygen optodes , 2001 .

[43]  J. Serôdio,et al.  Microphytobenthos vertical migratory photoresponse as characterised by light-response curves of surface biomass , 2006 .

[44]  B. Jørgensen,et al.  The light field of microbenthic communities: Radiance distribution and microscale optics of sandy coastal sediments , 1994 .

[45]  Robert A. Maffione,et al.  Effects of microalgal communities on reflectance spectra of carbonate sediments in subtidal optically shallow marine environments , 2003 .

[46]  B. Jørgensen,et al.  Contribution of Chloroflexus respiration to oxygen cycling in a hypersaline microbial mat from Lake Chiprana, Spain. , 2007, Environmental microbiology.

[47]  U. Witte,et al.  Effect of advective pore water transport on distribution and degradation of diatoms in permeable North Sea sediments , 2004 .

[48]  M. Pinkerton,et al.  Field spectrometry: New methods to investigate epilithic micro-algae on rocky shores , 2005 .

[49]  B. Jørgensen,et al.  The diffusive boundary layer of sediments: oxygen microgradients over a microbial mat. , 1990, Limnology and oceanography.

[50]  J. Waterbury,et al.  Generic assignments, strain histories, and properties of pure cultures of cyanobacteria , 1979 .

[51]  W. Smith,et al.  Culture of Marine Invertebrate Animals , 1975, Springer US.

[52]  M. Huettel,et al.  Rapid wave-driven advective pore water exchange in a permeable coastal sediment , 2004 .

[53]  S. Sathyendranath,et al.  Effect of pigment composition on absorption properties of phytoplankton , 1991 .

[54]  B. Jørgensen,et al.  Adaptation to Hydrogen Sulfide of Oxygenic and Anoxygenic Photosynthesis among Cyanobacteria , 1986, Applied and environmental microbiology.

[55]  H. Gemerden.  Microbial mats: A joint venture , 1993 .

[56]  Lubos Polerecky,et al.  Two-dimensional mapping of photopigment distribution and activity of Chloroflexus-like bacteria in a hypersaline microbial mat. , 2008, FEMS microbiology ecology.

[57]  B. Jørgensen,et al.  Diurnal Cycle of Oxygen and Sulfide Microgradients and Microbial Photosynthesis in a Cyanobacterial Mat Sediment , 1979, Applied and environmental microbiology.

[58]  F. E. Round,et al.  PERSISTENT, VERTICAL-MIGRATION RHYTHMS IN BENTHIC MICROFLORA. VI. THE TIDAL AND DIURNAL NATURE OF THE RHYTHM IN THE DIATOM HANTZSCHIA VIRGATA , 1967 .

[59]  M. Huettel,et al.  Advective pore‐water exchange driven by surface gravity waves and its ecological implications , 2003 .

[60]  J. Lawrence,et al.  Three-dimensional differentiation of photo-autotrophic biofilm constituents by multi-channel laser scanning microscopy (single-photon and two-photon excitation). , 2004, Journal of microbiological methods.

[61]  F. E. Round,et al.  Persistent, vertical-migration rhythms in benthic microflora , 1965 .

[62]  B. Jørgensen,et al.  Bacterial zonation, photosynthesis, and spectral light distribution in hot spring microbial mats of Iceland , 1988, Microbial Ecology.

[63]  F. Sansone,et al.  Christmas Island lagoonal lakes, models for the deposition of carbonate{evaporite{organic laminated sediments , 2001 .

[64]  F. Shiraishi,et al.  Photosynthesis, Respiration and Exopolymer Calcium-Binding in Biofilm Calcification (Westerhöfer and Deinschwanger Creek, Germany) , 2008 .

[65]  F. E. Round,et al.  Persistent, vertical-migration rhythms in benthic microflora.: II. Field and Laboratory Studies On Diatoms From The Banks Of The River Avon , 1966, Journal of the Marine Biological Association of the United Kingdom.

[66]  T. Neu,et al.  An Endolithic Microbial Community in Dolomite Rock in Central Switzerland: Characterization by Reflection Spectroscopy, Pigment Analyses, Scanning Electron Microscopy, and Laser Scanning Microscopy , 2006, Microbial Ecology.

[67]  G. Underwood,et al.  THE UPS AND DOWNS OF LIFE IN A BENTHIC BIOFILM: MIGRATION OF BENTHIC DIATOMS , 2004 .

[68]  M. Kühl,et al.  Regulation of photosynthesis and oxygen consumption in a hypersaline cyanobacterial mat (Camargue, France) by irradiance, temperature and salinity. , 2006, FEMS microbiology ecology.

[69]  M. Roldán,et al.  Noninvasive Pigment Identification in Single Cells from Living Phototrophic Biofilms by Confocal Imaging Spectrofluorometry , 2004, Applied and Environmental Microbiology.

[70]  U. Werner,et al.  Surficial and deep pore water circulation governs spatial and temporal scales of nutrient recycling in intertidal sand flat sediment , 2006 .

[71]  J. Serôdio,et al.  NONDESTRUCTIVE TRACING OF MIGRATORY RHYTHMS OF INTERTIDAL BENTHIC MICROALGAE USING IN VIVO CHLOROPHYLL A FLUORESCENCE 1, 2 , 1997 .

[72]  E. Govindje,et al.  Sixty-Three Years Since Kautsky: Chlorophyll a Fluorescence , 1995 .

[73]  H. Gemerden,et al.  Growth of the cyanobacterium Microcoleus chtonoplastes on sulfide , 1988 .

[74]  L. Stal.  Physiological ecology of cyanobacteria in microbial mats and other communities. , 1995, The New phytologist.

[75]  Govindjee,et al.  Chlorophyll a Fluorescence: A Bit of Basics and History , 2004 .

[76]  F. Colijn,et al.  Phytoplankton monitoring by flow cytometry , 1994 .

[77]  E. Rosenberg,et al.  Microbial mats : physiological ecology of benthic microbial communities , 1989 .

[78]  C. Sotin,et al.  Mapping microphytobenthos biomass by non-linear inversion of visible-infrared hyperspectral images , 2005 .

[79]  M. Kühl,et al.  Artificial Cyanobacterial Mats: Growth, Structure, and Vertical Zonation Patterns , 2000, Microbial Ecology.

[80]  Li Yang,et al.  Revised Kubelka-Munk theory. III. A general theory of light propagation in scattering and absorptive media. , 2005, Journal of the Optical Society of America. A, Optics, image science, and vision.

[81]  Erwin Mayer,et al.  Improved Curve Resolution of Highly Overlapping Bands by Comparison of Fourth-Derivative Curves , 1996 .

[82]  J. Kromkamp,et al.  Determination of microphytobenthos PSII quantum efficiency and photosynthetic activity by means of variable chlorophyll fluorescence , 1998 .

[83]  Karen Helen Wiltshire,et al.  Microbiological mediation of spectral reflectance from intertidal cohesive sediments , 1998 .

[84]  J. Waterbury,et al.  Discrimination between types of pigments in marine Synechococcus spp. by scanning spectroscopy, epifluorescence microscopy, and flow cytometry1 , 1985 .

[85]  F. E. Muller-Karger,et al.  Multi-scale remote sensing of microbial mats in an atoll environment , 2003 .

[86]  R. Airs,et al.  A high resolution study of the chlorophyll and bacteriochlorophyll pigment distributions in a calcite/gypsum microbial mat , 2003 .

[87]  D. de Beer,et al.  Spatial and temporal patterns of mineralization rates and oxygen distribution in a permeable intertidal sand flat (Sylt, Germany) , 2006 .

[88]  J. Cullen,et al.  FLUORESCENCE‐BASED MAXIMAL QUANTUM YIELD FOR PSII AS A DIAGNOSTIC OF NUTRIENT STRESS , 2001 .

[89]  H. Tamiaki,et al.  Fluorescence spectroscopy of single photosynthetic light-harvesting supramolecular systems , 2007, Cell Biochemistry and Biophysics.