Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms

A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F(420)-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.     

Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere: new perspectives for studying microbial communities

To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.     

Tracking the autochthonous carbon transfer in stream biofilm food webs

Food webs in the rhithral zone rely mainly on allochthonous carbon from the riparian vegetation. However, autochthonous carbon might be more important in open canopy streams. In streams, most of the microbial activity occurs in biofilms, associated with the streambed. We followed the autochthonous carbon transfer toward bacteria and grazing protozoa within a stream biofilm food web. Biofilms that developed in a second-order stream (Thuringia, Germany) were incubated in flow channels under climate-controlled conditions. Six-week-old biofilms received either 13C- or 12C-labeled CO?, and uptake into phospholipid fatty acids was followed. The dissolved inorganic carbon of the flow channel water became immediately labeled. In biofilms grown under 8-h light/16-h dark conditions, more than 50% of the labeled carbon was incorporated in biofilm algae, mainly filamentous cyanobacteria, pennate diatoms, and nonfilamentous green algae. A mean of 29% of the labeled carbon reached protozoan grazer. The testate amoeba Pseudodifflugia horrida was highly abundant in biofilms and seemed to be the most important grazer on biofilm bacteria and algae. Hence, stream biofilms dominated by cyanobacteria and algae seem to play an important role in the uptake of CO? and transfer of autochthonous carbon through the microbial food web.     

Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy

Recent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light. We have measured the two-photon excitation spectra and cross sections of the green and the red species as well as the spectral dependence of two-photon conversion. The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. Optical highlighting of small subcellular compartments was shown on HeLa cells expressing EosFP fused to a mitochondrial targeting signal. After three-dimensionally confined two-photon conversion of EosFP within the mitochondrial networks of the cells, the converted regions could be resolved in a 3D reconstruction from a dual-color 4Pi image stack.     

Immunofluorescence analysis in flow cytometry: better selection of antibody-labeled cells after fluorescence overcompensation in the red channel

Selection of cells labeled with fluorescein isothiocyanate-conjugated (FITC) antibodies can be difficult if large autofluorescent cells are used and if the cells bind only a few molecules of antibody. We have developed a simple flow cytometric procedure that allows better selection of stained cells. When an argon ion laser emitting at 488 nm is used, the green fluorescence detected is the sum of cell autofluorescence and of the signal generated by the FITC antibody. Thus, when we subtract green signal from the red by fluorescence compensation, the signal of stained cells is on average reduced more than for the unstained counterpart. In this scenario, positive selection of cells with low red signal allows more efficient selection of stained cells. We tested the overcompensation procedure on mixtures of cells unstained and stained with a relevant FITC antibody. Cell mixtures were analyzed using normal vs increased levels of compensation in the red channel. Increased levels of compensation resulted in easier gating and higher recovery of stained cells. The efficiency of the overcompensation procedure was particularly high when using red filters with low cutoff (i.e., 560 or 570 nm), possibly because of the significant emission of fluorescein in the red channel, which caused separation between stained and unstained cells also in the red dimension. This method is useful for sorting cells expressing low levels of surface markers and facilitates selection of rare cells transfected with surface antigen genes. This technique is compatible with the use of propidium iodide for live/dead cell discrimination and with the subtraction of the cellular background of autofluorescence.     

SPECTRAL FINGERPRINTING OF ALGAL COMMUNITIES: A NOVEL APPROACH TO BIOFILM ANALYSIS AND BIOMONITORING1

A new technique for spectral fingerprinting of major algal groups in the freshwater periphyton (i.e. cyanobacteria, green algae, and diatoms) was developed using confocal laser scanning microscopy. This technique used the differential spectral emission signatures of photosynthetic algae and allowed their spatially explicit quantification and community three‐dimensional reconstruction. Algal biovolume measurements, carried out with this technique, are superior to existing protocols involving chl and ash‐free dry mass assessments because they are nondestructive, localized, and specific at a group level. This technique can be used to generate depth profiles of the periphytic mat with various applications in aquatic ecology and biofilm analysis.     

Development and dynamics of Pseudomonas sp. biofilms

Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.     

One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos

We constructed a photoactivatable Drosophila histone 2 A variant green fluorescent fusion protein (H2AvD-paGFP) for tracking chromatin loci in living Drosophila embryos. Activation of paGFP was achieved by irradiation from a single-photon diode laser at 408 nm, but activated nuclei failed to divide. Photoconversion could also be achieved by two-photon fs pulses in the range of 780-840 nm. Viability in whole-mount embryos could only be maintained at 820 nm, at which we could activate, simultaneously track and quantitate the mobility of multiple fluorescent loci. This report constitutes the first demonstration of two-photon activation of paGFP and the use of a paGFP-fusion protein in investigations of whole organisms.     

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

Halite deposits from the hyperarid zone of the Atacama Desert reveal the presence of endolithic microbial colonization dominated by cyanobacteria associated with heterotrophic bacteria and archaea. Using the λ-scan confocal laser scanning microscopy (CLSM) option, this study examines the autofluorescence emission spectra produced by single cyanobacterial cells found inside halite rocks and by their photosynthetic pigments. Photosynthetic pigments could be identified according to the shapes of the emission spectra and wavelengths of fluorescence peaks. According to their fluorescence fingerprints, three groups of cyanobacterial cells were identified within this natural extreme microhabitat: (i) cells producing a single fluorescence peak corresponding to the emission range of phycobiliproteins and chlorophyll a, (ii) cells producing two fluorescence peaks within the red and green signal ranges, and (iii) cells emitting only low-intensity fluorescence within the nonspecific green fluorescence signal range. Photosynthetic pigment fingerprints emerged as indicators of the preservation state or viability of the cells. These observations were supported by a cell plasma membrane integrity test based on Sytox Green DNA staining and by transmission electron microscopy ultrastructural observations of cyanobacterial cells.     

兰州五泉山的藻类及其分布

以兰州五泉山为该地藻种资源库,对其中水生、陆生生境中藻类的种类多样性、群落结构、分布特点进行了研究。结果发现该地藻类植物65种(含4变种),包括蓝藻、绿藻、硅藻和红藻,其中硅藻种类最多(29种),其它依次为蓝藻(24种)、绿藻(11种)和红藻(1种)。水体中共42种,硅藻最多,有26种,其次蓝藻8种,绿藻7种,红藻1种,不同水体中优势种和亚优势种不同。土壤生境中发现20种,蓝藻13种,绿藻4种,硅藻3种,且非洲席藻和小球藻分为优势种和亚优势种。7个种类在水、陆两大生境都有分布,而且它们主要是丝状蓝藻。     

Early stages of biofilm succession in a lentic freshwater environment

Initial events of biofilms development and succession were studied in a freshwater environment at Kalpakkam, East Coast of India. Biofilms were developed by suspending Perspex (Plexiglass) panels for 15 days at bimonthly intervals from January 1996 to January 1997. Changes in biofilm thickness, biomass, algal density, chlorophyll a concentration and species composition were monitored. The biofilm thickness, biomass, algal density and chlorophyll a concentration increased with biofilms age and colonization was greater during summer (March, May and July) than other months. The initial colonization was mainly composed of Chlorella vulgaris, Chlorococcum humicolo (green algae), Achnanthes minutissima, Cocconeis scutellum, C. placentula (diatoms) and Chroococcus minutus (cyanobacteria) followed by colonial green algae such as Pediastrum tetras, P. boryanumand Coleochaete scutata, cyanobacteria (Gloeocapsa nigrescens), low profile diatoms (Amphora coffeaeformis, Nitzschia amphibia, and Gomphonema parvulum) and long stalked diatoms (Gomphoneis olivaceumand Gomphonema lanceolatum). After the 10th day, the community consisted of filamentous green algae (Klebshormidium subtile, Oedogonium sp., Stigeoclonium tenue and Ulothrix zonata) and cyanobacteria (Calothrix elenkinii, Oscillatoria tenuis and Phormidium tenue). Based on the percentage composition of different groups in the biofilm, three phases of succession could be identified: the first phase was dominated by green algae, the second by diatoms and the third phase by cyanobacteria. Seasonal variation in species composition was observed but the sequence of colonization was similar throughout the study period.     

Confocal imaging of in situ natural microbial communities and their extracellular polymeric secretions using Nanoplast resin

A novel method using excision and fixation in Nanoplast, a hydrophilic embedding resin, allows confocal imaging of natural microbial communities and their extracellular polymeric secretions (EPS) while in situ. Prestaining with fluorescent probes permits the observation of specific cellular and extracellular components. Marine stromatolite sediments were examined using this method. Optical sectioning using confocal laser scanning microscopy (CLSM) permitted high-resolution imaging through sediments. Delicate arrangements of the EPS that are associated with sedimentary microbial biofilms were imaged using a fluorescein isothiocyanate (FITC)-labeled lectin (concanavalin-A) probe. Close microspatial associations of heterotrophic bacteria cells and autotrophic cyanobacteria cells were also observed. The nanoplast resin produces no detectable autofluorescence. Further coupling of multi-photon scanning laser microscopy (2P-LSM) with a conventional single photon CLSM allowed concurrent imaging of DAPI-labeled microbial cells, FITC-labeled EPS and autofluorescent carbonate sand grains. The multi-photon infrared laser permits deep (approximately 1 mm) penetration of samples and the excitation of DAPI, which normally requires UV-excitation with minimal disturbance to samples. The unique combination of Nanoplast with fluorescent probes, CLSM and 2P-LSM allows for the preservation and imaging of natural microbial communities in their in situ state, a method easily adapted for examinations of other microbial systems.     

DNA content of Ulva compressa (Ulvales, Chlorophyta) nuclei determined with laser scanning cytometry

The green macroalgal genus Ulva (incl. Entemmorpha) contains economically valuable species, is of relevance for coastal management (green tides), and certain taxa serve as experimental organisms for fundamental research in green algae. The nuclear genome size of Ulva (Entemmorpha) compressa Linnaeus was measured in propidium iodide stained nuclei using laser scanning cytometry. Nuclei of fixed gametes yielded reproducible values, whereas nuclei extracted from multicellular gametophytes were unsuitable. With nuclei of Arabidopsis thaliana (L.) Heynh and Saccharomyces cerevisiae Hansen as references, the haploid nuclear genome size of U. compressa was calculated as 135 ± 7 Mbp. This is the smallest genome so far known from any species of Ulva.     

A Color Image Analysis Method for Assessment of Germination Based on Differential Fluorescence Staining of Bacterial Spores and Vegetative Cells Using Acridine Orange

Color fluorescence image analysis of acridine orange (AO) stained germinating Bacillus subtilis var. niger bacteria revealed a cell population initially dominated by small green spores followed by the emergence of at least three additional discernible subpopulations in response to stimulation with D-glucose. These subpopulations were small, round or oblong red cells; intermediate to large metachromatic cells; and large red rods. Large green rods were rarely observed. An increase in red emissions (i.e., putative RNA synthesis) was sometimes seen as early as 90 min after exposure to D-glucose and uptake of AO at room temperature. This may represent either metabolic recovery from quiescence or RNA synthesis associated with germination. In the absence of D-glucose, or using autoclaved bacteria in the presence of glucose, no relative increase in the red signal was observed despite hours of observation. Digital image analysis was used for relative measurement of red, green and blue signals and to correlate the size of various subpopulations with their fluorescence color emissions over time. Image analysis demonstrated a trend toward increasing size and red emission in the presence of glucose. The average red emission was found to be a good discriminator of the various subpopulations, while the average green emission was approximately equal among the subpopulations making it a poor discriminator. These data suggest that AO staining might be used for rapid computer-assisted discrimination of spores vs. vegetative cells.     

可用于双色双光子显微成像的新的荧光蛋白对

双色双光子激光扫描显微技术可以用来研究生物组织内两种不同蛋白质的表达、定位和示踪．由于大多数双光子显微镜一次只能提供一种波长的激发光,双色同时成像较难实现．mAmetrine和mKate2作为新发现的荧光蛋白对可以用于双光子双色同时成像,这得益于它们各自的优势：mAmetrine的斯托克斯位移和mKate2的高亮度．在765nm的波长激发时,它们的双光子吸收效率都很高．mAmetrine和mKate2能够很好地用于双色双光子活细胞成像实验．     

Patterns of Biofilm Succession on a Sheltered Rocky Shore in Hong Kong

Successional patterns are dependent on the nature of the substratum, water flow, concentrations of organics as well as the availability of bacteria, algal spores and invertebrate larvae in the coastal environment. Bacteria play an especially important role in biofilm formation as they are generally the earliest colonizers. In the present study, both winter and summer biofilm succession patterns were examined on glass coverslips inverted on experimental racks attached at two tidal levels on a sheltered shore in Hong Kong. In the succession, bacteria were followed by diatoms and cyanobacteria. Encrusting algae appeared in the late stages of the experiment (day 80 in summer and day 60 in winter). Colonization by bacteria was much slower in summer and their density remained low throughout the experimental period. The first appearance of diatoms and cyanobacteria, however, was more rapid in the summer. Bacteria and diatoms on the low-shore surfaces also had a faster succession rate than on the high-shore surfaces, suggesting that desiccation/aerial temperature are the causal factors for such differences.     

Patterns of biofilm succession on a sheltered rocky shore in Hong Kong

Successional patterns are dependent on the nature of the substratum, water flow, concentrations of organics as well as the availability of bacteria, algal spores and invertebrate larvae in the coastal environment. Bacteria play an especially important role in biofilm formation as they are generally the earliest colonizers. In the present study, both winter and summer biofilm succession patterns were examined on glass coverslips inverted on experimental racks attached at two tidal levels on a sheltered shore in Hong Kong. In the succession, bacteria were followed by diatoms and cyanobacteria. Encrusting algae appeared in the late stages of the experiment (day 80 in summer and day 60 in winter). Colonization by bacteria was much slower in summer and their density remained low throughout the experimental period. The first appearance of diatoms and cyanobacteria, however, was more rapid in the summer. Bacteria and diatoms on the low-shore surfaces also had a faster succession rate than on the high-shore surfaces, suggesting that desiccation/aerial temperature are the causal factors for such differences.