Reflectance spectroscopy and laser confocal microscopy as tools in an ecophysiological study of microbial mats in an alpine bog pond

Wiggli, Markus; Smallcombe, Anna; Bachofen, Reinhard

doi:10.1016/s0167-7012(98)00085-2

Cited by 27 publications

(26 citation statements)

References 34 publications

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“…The 488-, 514-, and 543-nm exc , absorbed essentially by C-phycoerythrin (C-PE), resulted in strong orange fluorescence emission at 579 Ϯ 1.6 nm in vivo. These results are consistent with those previously reported (12,16,15).…”

supporting

confidence: 94%

“…Fluorescence allows the description of a complex community in terms of physiological state (17), discrimination among phylogenetic groups (8), quantification of biomass (2), energy transfer, and cell evolution (4). Approaches to improve the use of cellular fluorescence techniques with subsequent localization of the organisms in two dimensions (2D) or 3D (15,10) have been reported.…”

mentioning

confidence: 99%

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Noninvasive Pigment Identification in Single Cells from Living Phototrophic Biofilms by Confocal Imaging Spectrofluorometry

Roldán¹,

Thomas

Castel

et al. 2004

Appl Environ Microbiol

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A new imaging technique for the analysis of fluorescent pigments from a single cell is reported. It is based on confocal scanning laser microscopy coupled with spectrofluorometric methods. The setup allows simultaneous establishment of the relationships among pigment analysis in vivo, morphology, and three-dimensional localization inside thick intact microbial assemblages.Phototrophic organisms produce a number of photosynthetic pigments that act as photoactivated fluorescent markers that switch on in response to light at a particular wavelength. A fraction of the energy absorbed by pigments may be emitted immediately at a longer wavelength, which is known as fluorescence (7). Fluorescence allows the description of a complex community in terms of physiological state (17), discrimination among phylogenetic groups (8), quantification of biomass (2), energy transfer, and cell evolution (4). Approaches to improve the use of cellular fluorescence techniques with subsequent localization of the organisms in two dimensions (2D) or 3D (15, 10) have been reported.Here we propose a new application of the confocal scanning laser microscope (CSLM) coupled with a spectrofluorometric detector. Confocal imaging spectrofluorometry provides simultaneous 3D information on photosynthetic microorganisms and their fluorescence signatures within thick assemblages (as in microbial mats, biofilms, etc.) because of their multiple excitation wavelengths ( exc s) and free detection of emitted wavelengths. We report our results on pigments, pure cultures, and biofilms from dim-light environments.Pigments. The pure pigments chlorophyll a (Chl a) and Chl b, xanthophyll (Xant), R-phycoerythrin, allophycocyanin-XL (APC-XL), and C-phycocyanin (C-PC), all obtained from Sigma-Aldrich (St. Louis, Mo.), were used as controls. Pigment solutions were set at a final concentration of 1 mg/ml, and scans were done as described below.Culture and biofilm preparation. Two cultures of Nostoc humifusum Carm. (Cyanobacteria) and Muriellopsis sp. (Chlorophyta) at distinct stages of growth (exponential phase, 1 week; stationary phase, 3 weeks) and three stratified aerophytic biofilms from dim habitats, BF1, BF2, and BF3, which are described and identified elsewhere (6), were tested. The biofilms contained several photosynthetic phylogenetic groups (Cyanobacteria, Chlorophyta, and Bacillariophyta).CSLM. CSLM was performed with a Leica TCS-SP2 (Leica Microsystems, Mannheim, Germany). The wavelengths of the excitation lasers were in the UV Ar (351 and 364 nm), blue Ar (458, 476, and 488 nm), green Ar (514 nm), green HeNe (543 nm), and red HeNe (633 nm) spectra. Each image sequence (wavelength scans or lambda scan function of the system) was obtained by scanning the same x-y optical section with a step size of 20 nm for emission wavelengths between 360 and 800 nm. The x, y, data set was acquired at the z position at which the fluorescence was maximal, and acquisition settings were not altered throughout the experiments. The variation in intensity of a particular spec...

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supporting

confidence: 94%

mentioning

confidence: 99%

Noninvasive Pigment Identification in Single Cells from Living Phototrophic Biofilms by Confocal Imaging Spectrofluorometry

Roldán¹,

Thomas

Castel

et al. 2004

Appl Environ Microbiol

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“…followed by more complex, filamentous and ramified sessile forms. A true layering of the cyanobacterial biofilm fraction was not observed, but it must be stressed that filamentous cyanobacteria are capable of vertical movements in microbial aggregates in response to a range of environmental stimuli, especially light (Wiggli et al 1999). When the phototrophic community was poorly diverse and dominated by filamentous cyanobacteria, as in February 2002, there was no observed vertical stratification in microbial distribution.…”

Section: Cyanobacteriamentioning

confidence: 94%

Seasonal succession of phototrophic biofilms in an Italian wastewater treatment plant: biovolume, spatial structure and exopolysaccharides

Congestri

Pippo²,

Philippis³

et al. 2006

Aquat. Microb. Ecol.

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A multiphasic approach was applied to investigate the structural features of phototrophic biofilms that grow in a wastewater treatment plant (WWTP) at Fiumicino Airport (Rome, Italy). Seasonal variations in species composition, biomass and exopolysaccharides produced were analyzed by light (LM) and electron microscopy (SEM), high performance liquid chromatography (HPLC) and circular dichroism (CD). Phototroph contribution to the 3-dimensional structure of the biofilm and its development was assessed by confocal laser scanning microscopy. Analysis of biofilms grown on polypropylene slides showed a stable species composition; seasonal changes in biomass were mostly due to changes of major cyanobacterial and algal taxonomic groups. Extensive growth was evident on the range of artificial substrata that were implanted in the treatment plant. CD spectra and HPLC analyses of 2 operationally defined exopolysaccharide fractions extracted from samples scraped off the tank walls revealed that negatively charged heteropolysaccharides comprised most of the matrix and capsular components of the biofilms. Cytochemical staining distinguished between acidic and sulphated residues in the samples observed by LM. The data provide a new insight into the structural integrity and development of phototrophic biofilms in this hyper-eutrophic environment, indicating a potential use of autochthonous consortia in an environmentally sound tertiary water treatment alternative to conventional chemico-physical technologies.KEY WORDS: Phototrophic biofilms · Exopolysaccharides · Cyanobacteria · Algae · Biovolume · Confocal laser scanning microscope · Wastewater treatment plant Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 45: [301][302][303][304][305][306][307][308][309][310][311][312] 2006 and with substrate adhesion (stalks, tubes) and locomotion in benthic forms (Hoagland et al. 1993). Many representatives of green algae possess cellulose or glycoprotein cell walls and capsules (van den Hoek et al. 1995, Shubert 2003, several colonial forms are embedded in mucilage matrix, while mucilage is extruded by desmids for gliding (Nultsch & Hader 1988). In addition, many planktic forms release dissolved organic material, which comprises a significant fraction of polysaccharides (Kaplan 1987, Lombardi & Vieria 1999, Lombardi et al. 2005.Overall, EPS are involved in adhesion to the substrata and cohesion between the cells of a biofilm; they play a role in surface-associated motility and provide protection against desiccation and grazing by predators, producing a matrix network that embeds cells and detritus (Wingender et al. 1999, Decho 2000. These exopolymers provide a microenvironment where UVabsorbing pigments and proteins, including enzymes, are immobilized (Sutherland 2001). The major components of the matrix are polysaccharides, but proteins and nucleic acids can comprise a significant part (Decho 2000). Extracellular polysaccharides may also serve in the immobilization and accumula...

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“…New insights have also been derived from the application of novel microscopic methods to these systems, including low-temperature electron microscopy (LTSEM) and confocal laser scanning microscopy (CLSM). These new technologies have shown that microbial mats are more dynamic and structured than previously thought (4,26,43).…”

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confidence: 98%

Microstructural Characterization of Cyanobacterial Mats from the McMurdo Ice Shelf, Antarctica

Rı́os

Ascaso

Wierzchos

et al. 2004

Appl Environ Microbiol

135

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The three-dimensional structures of two types of cyanobacterium-dominated microbial mats from meltwater ponds on the McMurdo Ice Shelf were as determined by using a broad suite of complementary techniques, including optical and fluorescence microscopy, confocal scanning laser microscopy, scanning electron microscopy with back-scattered electron-imaging mode, low-temperature scanning electron microscopy, and microanalyitical X-ray energy dispersive spectroscopy. By using a combination of the different in situ microscopic techniques, the Antarctic microbial mats were found to be structures with vertical stratification of groups of cyanobacteria and mineral sediments, high contents of extracellular polymeric substances, and large void spaces occupied by water. In cyanobacterium-rich layers, heterocystous nostocalean and nonheterocystous oscillatorialean taxa were the most abundant taxa and appeared to be intermixed with fine-size deposits of epicellular silica and calcium carbonate. Most of the cyanobacterial filaments had similar orientations in zones without sediment particles, but thin filaments were tangled among thicker filaments. The combination of the microscopic techniques used showed the relative positions of biological and mineral entities within the microbial mats and enabled some speculation about their interactions.

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Reflectance spectroscopy and laser confocal microscopy as tools in an ecophysiological study of microbial mats in an alpine bog pond

Cited by 27 publications

References 34 publications

Noninvasive Pigment Identification in Single Cells from Living Phototrophic Biofilms by Confocal Imaging Spectrofluorometry

Noninvasive Pigment Identification in Single Cells from Living Phototrophic Biofilms by Confocal Imaging Spectrofluorometry

Seasonal succession of phototrophic biofilms in an Italian wastewater treatment plant: biovolume, spatial structure and exopolysaccharides

Microstructural Characterization of Cyanobacterial Mats from the McMurdo Ice Shelf, Antarctica

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