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Microbial phototrophic communities in sediments and biofilms are often used as small-scale model systems in studies of microbial interaction (15). In the absence of bioturbation and grazing, such systems exhibit characteristic stratification of both microbial processes and different photosynthetic populations over micrometer to submillimeter distances. Microsensor techniques (8, 14) allow repetitive and nondestructive high-resolution measurement of physical parameters (light and temperature), chemical parameters (e.g., pH and O₂, CO₂, and H₂S contents), and metabolic rates (photosynthesis and respiration) in photosynthetic sediments and biofilms (5, 16). For the most part, analysis of the microdistribution of phototrophic microorganisms has relied on determining characteristic depth-dependent photopigment signatures by destructive techniques, such as horizontal slicing of a sample and subsequent pigment extraction and analysis (1, 10, 13), or by spectral absorbance and fluorescence microscopy of vertical thin sections obtained by freeze sectioning (4).

Nondestructive techniques have involved laser scanning confocal microscopy and reflectance spectroscopy of translucent biofilms, but the generally high optical densities of most sediments or microbial mats limit this approach to the upper 100 to 200 µm of samples (9, 17). Another approach is based on spectral measurement of the internal light fields of biofilms and sediments with fiber-optic microprobes, in which photopigment distributions are inferred from depth-dependent changes in spectral signatures for characteristic pigments (7, 11, 13). Besides reflectance and transmission spectroscopy, fiber-optic microprobes can also be used for microscale fluorescence analysis of biofilms and sediments (4). Here we describe a new fiber-optic fluorometer which can detect photopigments by their characteristic fluorescence as sampled via a fiber-optic microprobe.

Fiber-optic fluorometer. The technical concept of the new fluorometer is shown in Fig. 1. Ultrabright light-emitting diodes (LEDs) equipped with short-pass filters are used as excitation light sources. The excitation light is coupled into a single-strand step index silica glass fiber (core/cladding diameter, 100/140 µm) via a standard ST connector. The fiber is connected to a multimode 2×2 fused fiber coupler (Gould Inc.) (i.e., a fiber-optic beam splitter). One of the coupler branches is connected to the fiber-optic microprobe (see below). The excitation light illuminates the sample around the microprobe tip and excites the fluorophores present. A fraction of the induced diffuse fluorescent light reenters the glass fiber through the microprobe tip and is guided via the fiber coupler to a photomultiplier tube (PMT) (H5702-50; Hamamatsu). Removable light filters are positioned between the fiber end face and the PMT detector. The PMT provides a voltage signal proportional to the measured light intensity.

View larger version (52K): [in this window] [in a new window]   FIG. 1.   Technical concept of the fiber-optic fluorometer.

Fiber-optic microprobe. Fiber-optic microprobes were prepared as described by Kühl and Jørgensen (6) from a 100/140 µm (core/cladding ratio) step index fused silica fiber cable (numerical aperture, 0.22) with a standard ST plug at one end. The polyvinyl chloride tubing and protective plastic coating of the glass fiber were stripped for a length of approximately 40 mm, and the bare fiber was tapered in a small acetylene-oxygen flame to obtain a tip diameter of 5 to 10 µm. The taper was cut to obtain a fiber tip diameter of 50 µm. The tip was heated in a hot flame, which resulted in a rounded tip without sharp edges. For better handling the fiber-optic microprobe was fixed with epoxy resin in a hypodermic needle mounted on a syringe, which then was attached to a micromanipulator (Märtzhäuser GmbH).

Light sources and filter combinations. The fluorometer was configured with four ultrabright LEDs with the following peak wavelengths and half band widths, respectively: 470 and 20 nm (BP280CWPB1K; DCL Components), 515 and 35 nm (NSPE 510S; Nichia, Inc.), 590 nm (TLHE 5800; VISHAY), and 610 and 20 nm (L-513TUEC; DCL Components). The LEDs were combined with short-pass filters KP 480 and KP 540 (OIB GmbH), SP 600 (CVI), and KP 620 (OIB) with cutoff wavelengths of 480, 540, 600, and 620 nm, respectively. Below these filtered excitation light sources are referred to as blue, green, yellow, and orange, respectively.

Chlorophyll in liquid media. A pure liquid culture of the marine green alga Nannochloris sp. was diluted in seven steps by a factor two. At each dilution step the chlorophyll a fluorescence was measured with the blue  red+NIR (excitation  emission) combination. The chlorophyll a content of the undiluted culture was measured by a standard ethanol extraction method (12). A strong linear correlation (r² > 0.998) was found between the fluorescence signal and the chlorophyll concentration (Fig. 2). Even though the system was optimized for obtaining measurements in optically dense biofilms and sediments, it can also be used for direct quantitative measurement of chlorophyll in liquid media with reasonable sensitivity. Greater sensitivity in liquid media can be obtained, but this would involve modification of the sampling geometry at the fiber tip.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Chlorophyll a fluorescence in a liquid culture of Nannochloris sp. The error bars indicate standard deviations (n = 4). a. u., arbitrary units.

Fluorescence fingerprints. Fluorescence fingerprints of phototrophic microorganisms were obtained by placing the sensor tip in samples of five different species that were representative of different types of phototrophs (Fig. 3). Pure cultures of Euglena gracilis, Chromatium gracile (recently reclassified as Marichromatium gracile comb. nov. [3]), and Synechococcus sp. were centrifuged in a test tube (150 × g, 4 min). Subsequently, the fluorescence fingerprints inside the pellets obtained were determined. A sample of green sulfur bacteria was obtained from an enrichment culture in a Winogradsky column incubated for several months at 20°C in dim daylight. An in vivo absorption spectrum of the enrichment culture revealed that bacteriochlorophyll c was the dominant pigment. Pelagic diatoms were obtained from Øresund (Denmark) with a plankton net (mesh size, 10 µm). In order to avoid any influence of spatial heterogeneity, all excitation  emission values were determined in each sample without moving the sensor tip. The fluorescence signals were expressed as percentages of the blue  red+NIR fluorescence, which detected fluorescence from both chlorophylls and bacteriochlorophylls.

View larger version (81K): [in this window] [in a new window]   FIG. 3.   Fluorescence fingerprints of different phototroph species.

Measurements in artificial biofilms. In order to investigate the spatial resolution of the fiber-optic microprobe, we obtained measurements in defined biofilms of known composition and thickness. Three stratified layers consisting of 2% agar were prepared in a test tube. A homogenized suspension of Nannochloris sp. was added to the semisolid (~45°C) agar used for the middle layer. Fluorescence was measured in 200-µm vertical resolution steps with the blue  red+NIR combination (Fig. 4A). As the fiber tip approached the upper surface of the Nannochloris layer, the fluorescence signal increased exponentially from a distance 1 mm above that surface. About 100 µm above the interface the signal exhibited a maximum value, which was about two times higher than the average signal value inside the Nannochloris layer. When the fiber tip approached the lower limit of the Nannochloris layer at a depth of 5 mm, the signal decreased to one-half of the initial value within ~200 µm. The spatial resolution of a microscale fluorescence measurement is very dependent on the optical density of the sample in front of the fiber tip. At a low optical density (e.g., when measurements are obtained in agar), a large volume in front of the fiber tip is excited, which results in low spatial resolution, as shown by the increase in fluorescence 1 mm above the Nannochloris layer (Fig. 4B). At a higher optical density, a smaller volume is excited, which results in greater spatial resolution, as shown by the rapid decrease in the fluorescence signal at the lowermost boundary of the Nannochloris layer. It is important to keep these limitations of the technique in mind when fluorescence profiles are determined for natural samples like stratified microbial communities in sediments or microbial mats. However, in many cases the optical density is much greater than that of the Nannochloris layer described here and the spatial resolution is <100 to 200 µm (4). It is important to consider spatial resolution in samples with gelatinous layers of exopolymers with a low optical density comparable to that of agar.

View larger version (33K): [in this window] [in a new window]   FIG. 4.   Fluorescence microprofiles in artificial biofilms. (A) Nannochloris sp. embedded in an agar layer. (B) Multilayer system containing diatoms (depth, 0 to 2.3 mm) and green sulfur bacteria (depth, 2.3 to 5.5 mm). a.u., arbitrary units.

Application in a photosynthetic sediment. The system was used to study cyanobacterial distribution in a marine sulfidic sediment sample obtained with a core tube (diameter, 45 mm) in Øresund (Denmark) at a water depth of 6 m. The overlaying water in the core had a salinity of 22 ppt and was constantly aerated. The core was kept at 20°C in dim daylight before the experiment. Most of the sediment surface was covered with Beggiatoa sp. In the center of the surface layer, a circular area (diameter, ~15 mm) was covered by a dense 2-mm-thick layer of motile, large, filamentous cyanobacteria (Oscillatoria sp.). At the beginning of the experiment, additional illumination was applied with a halogen bulb (Philips 6423; 15 V; 150 W; distance, 20 cm), which resulted in an increase in the surface area covered by the cyanobacteria (Fig. 5D). The chlorophyll fluorescence was monitored over a transect in the upper 3 mm of the sediment. The transect data included five fluorescence profiles for the blue  red+NIR combination (200-µm depth intervals) for points that were 2 mm from each other. The transect data, which were acquired just before the illumination was switched on, are shown as a contour plot in Fig. 5A. The chlorophyll distribution showed that only the leftmost 4 mm of the sediment transect was covered by cyanobacteria. The transect values for the next 2 days (Fig. 5B and C) show the increase in the area covered by cyanobacteria. The measurements obtained show the potential of the new system for monitoring dynamic changes in the distribution of benthic phototrophic microorganisms due to migration.

View larger version (63K): [in this window] [in a new window]   FIG. 5.   Distribution of Oscillatoria sp. in a marine sulfidic sediment core for three consecutive days. Additional illumination was applied at zero time. (A to C) Transects of chlorophyll a fluorescence. (D) Sediment surface area covered by Oscillatoria sp. (top view). The positions of the profile sites are indicated by open circles. a.u., arbitrary units; t, time; d, day.

Application in intertidal sediment. Zonation of diatoms and cyanobacteria was investigated with intertidal sediments from the Rhone delta (Camargue, France). The sediment was dominated by diatoms overgrown by a thin layer of filamentous cyanobacteria. Numerous fluorescence microprofiles were obtained in a 100-mm² area. Figure 6 shows profiles obtained for all excitation  emission combinations. Vertical freeze sectioning and subsequent epifluorescence microscopy revealed two distinct layers: an upper layer ~300 µm thick dominated by filamentous cyanobacteria and a second layer 1.3 to 1.7 mm thick dominated by diatoms. The combined biomass of phototrophs was much greater in the upper layer than in the lower layer. Anoxygenic phototrophic bacteria were not detected by microscopy, and consequently the blue  NIR combination resulted in no significant fluorescence signal in the sediment. The yellow  orange and orange  red+NIR combinations revealed a high level of phycobilins over a depth interval which was similar to the measured thickness of the cyanobacterial top layer. The profiles obtained with the blue  red+NIR and green  red+NIR combinations also showed peaks in the top layer, but high signal values were also obtained for deeper layers where diatoms dominated.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Fluorescence microprofiles for different excitation  emission combinations in an intertidal sediment from the Rhone delta (Camargue, France). The error bars indicate standard deviations (n = 5). a.u., arbitrary units.

Summary. It is now possible to obtain microscale fluorescence measurements with fiber-optic microprobes for nondestructive mapping of photopigments in biofilms, sediments, and other surface-associated microbial communities. Microscale fluorescence measurements can also be used for quantitative measurement of pigment concentrations in liquid cultures. However, quantitative measurements cannot be obtained for optically dense samples, like sediments or microbial mats, without knowledge of the spatially heterogeneous absorption and scattering properties of the samples.