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

Abstract

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...

Fluorescent Fingerprints of Endolithic Phototrophic Cyanobacteria Living within Halite Rocks in the Atacama Desert

“…The emission spectra of cyanobacterial pigments were obtained using a wavelength -scan function of the CLSM based on a fluorescence method that determines the complete spectral distribution of the fluorescence signals emitted (20). Images were acquired with the same CLSM and objective.…”

“…Significant progress is constantly being made in detectors and computer technology, image analysis, visualization, laser sources, and optical technologies. These developments have allowed the noninvasive detection of photosynthetic pigment changes occurring in microorganisms in their natural environment (18,20).…”

Functional and structural imaging of phototrophic microbial communities and symbioses

“…2006), phototrophic biofilms and single cells (e.g. Neu et al 2004, Roldán et al 2004, Larson & Passy 2005Vermaas et al 2008), we are not aware of any micrometre to millimetre scale application of HS imaging in aquatic microbiology beyond our own recent research. The following will thus mainly give a brief introduction to our current HS imaging methodology.…”

“…Leica SP5 or similar microscopes) have aided a number of laboratory studies investigating the complex 3-dimensional structure of cells in natural phototrophic biofilms (Roldán et al 2004, Larson & Passy 2005 and in coral-associated bacterial communities (Ainsworth et al 2006). Such systems are under rapid development and, most recently, even sub-cellular pigment distributions within small unicellular cyanobacteria have been resolved (Vermaas et al 2008).…”

Ecology, morphology and physiology ofChroothece richteriana(Rhodophyta, Stylonematophyceae) in the highly calcareous Río Chícamo, south-east Spain

“…The wavelengths of the excitation lasers were in the UV Ar (351, 364 nm), blue Ar (458, 476, 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 λ-step size of 20 nm for emission wavelengths between 360 and 800 nm (Roldán et al, 2004). Colonies for scanning electron microscopy (SEM) were fixed in the field with glutaraldehyde and paraformaldehyde in cacodylate buffer, and subsequently postfixed with OsO4; they were then critical-point dehydrated and coated with gold-palladium before observations with a JEOL-6100 (JEOL, Tokyo, Japan).…”