In situ analysis of microbial consortia in activated sludge using fluorescently labelled,rRNA-targeted oligonucleotide probes and confocal scanning laser microscopy

Activated sludge flocs are complex consortia of various micro-organisms. The community structures of samples taken from municipal sewage treatment plants were characterized using fluorescently labelled, 16S and 23S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM). In comparison with conventional epifluorescence microscopy, CSLM considerably improved the capability to visualize directly the spatial distribution of defined bacterial populations inside the sludge flocs. Analyses could be performed at high resolution undisturbed by problems such as autofluorescence or limited spatial resolution in thick samples. In addition, CSLM was used to analyse some structural properties of paraformaldehyde-fixed activated sludge flocs, such as floc size and homogeneity. Typical floc sizes were found to be in the range between 5 and 50 μm. Whereas most of the flocs were completely colonized by bacteria, there were also examples of flocs containing gas bubbles or particles in the interior.     

Determination of the water droplet size distribution of fat spreads using confocal scanning laser microscopy

Knowledge of the water droplet size distribution of fat spreads is necessary for the development and production of high quality microbiological safe products. Fat spreads are water‐in‐oil emulsions. The water droplet size distribution can be determined by confocal scanning laser microscopy (CSLM) after staining the fat with Nile Red. The profiles of the non‐fluorescent water droplets in the 2D images are identified and measured using image analysis. The ‘true’ water droplet size distribution is calculated from the distribution of the measured profile diameters using a Wicksell transformation of log‐normal distributions. The influence of the fluorescent staining and CSLM parameters on the information were studied. The CSLM method was tested on fat spreads with a fat content ranging from 40% to 80%. The results were compared with those obtained by nuclear magnetic resonance spectroscopy (NMR). The distribution parameters [volume weighed geometric mean diameter (D?_3,3) and the standard deviation (σ) of the logarithm of the droplet diameter] calculated for 80% fat spreads are in good agreement with those obtained by NMR (within ± 7% relative). Small differences were found for 65% fat spreads and large differences were identified for 40% fat spreads. The precision for the determination of the D?_3,3 value by CSLM is worse than that of NMR, even when three images were used to calculate this parameter [3–11% and 1–6% relative standard deviation (RSD), respectively]. The precision for the determination of exp(σ) by CSLM is comparable or better than that of NMR (1–5% and 3–6% RSD, respectively). CSLM proved to be a reliable method for the determination of the water droplet size distribution of margarines (80% fat). The advantage of CSLM compared to NMR is that visual information is given about the water droplet size distribution in the sample.     

Dynamic confocal scanning laser microscopy methods for studying milk protein gelation and cheese melting

Dynamic confocal scanning laser microscopy (CSLM) methods were developed to enable observation of milk protein gelation and cheese melting. Protein aggregation and the formation of gel networks in renneted full-fat and low-fat milks and glucono-δ-lactone (GDL)-acidified skim milks were observed by CSLM and observations correlated with increases in shear modulus (G′) and dynamic viscosity (η*) as determined by dynamic amplitude oscillatory rheology. Confocal scanning laser microscopy observation of low-fat and full-fat cheeses showed changes in fat distribution and an increase in staining intensity during cheese melting.     

Registration of confocal scanning laser microscopy and quantitative backscattered electron images for the temporospatial quantification of mineralization density in 18-month old thoroughbred racehorse articular calcified cartilage

Combined backscattered electron scanning electron microscopy (BSE SEM) and confocal scanning laser microscopy (CSLM) have been used to put tissue mineralization data into the context of soft tissue histology and fluorescent label information. Mineralization density (Dm) and linear accretion rate (LAR) are quantifiable parameters associated with mineralizing fronts within calcified tissues. Quantitative BSE (qBSE) may be used to determine Dm, while CSLM may be used to detect label fluorescence from which LAR is calculated. Eighteen-month old Thoroughbred horses received single calcein injections 19 and 8 days prior to euthanasia, labeling sites of active mineralization with fluorescent bands. Confocal scanning laser microscopy images of articular calcified cartilage (ACC) from distal third metacarpal condyles were registered to qBSE images of the same sites using an in-house program. ImageJ and Sync Windows enabled the simultaneous collection of LAR and Dm data. The repeatability of the registration and measurement protocols was determined. Dm profiles between calcein labels were explored for an association with time. Dm was 119.7 +/- 24.5 (mean +/- standard deviation) gray levels (where 0 = backscattering from monobrominated and 255 from monoiodinated dimethacrylate standards, respectively), while modal and maximum LAR were 0.45 and 3.45 microm/day, respectively. Coefficients of variation (CV) for Dm were 0.70 and 0.77% with and without repeat registration, respectively; CVs for LAR were 1.90 and 2.26% with and without repeat registration, respectively. No relationship was identified between Dm and time in the 11-day interlabel interval. Registration of CSLM to qBSE images is sufficiently repeatable for quantitative studies of equine ACC.     

Radon track imaging in CR-39 plastic detectors using confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) has been used to provide the first images of radon track populations in two external CR-39 plastic detectors. Measurements of variables including track area distribution and estimates of the angle of track inclination (dip) derived from surface CSLM sections are presented. CSLM depth slices, combined with three-dimensional (3D) visualization techniques, provide a new, non-destructive way of examining the 2D and 3D geometry of the etched tracks within solid-state nuclear track detectors that may prove useful in complementing existing optical microscopy methods.     

Determination of localized diffusion coefficients in gels using confocal scanning laser microscopy

The development of a photobleaching technique, CFMM (continuous fluorescence multipoint microphotolysis), to measur e diffusion coefficients in gel systems using a confocal scanning laser microscope is described. Diffusion coefficients (D) were determined for fluorescently labelled dextrans of varying molecular weight in agarose gels, and the results compared with two other methods. CFMM enabled diffusion coefficients to be rapidly determined from the profile across an irradiated area within a defined microscopic location of the gel. The technique was experimentally simple and produced values of D that corresponded well with classical double-diffusion cell methods.     

Confocal laser microscopy and three-dimensional reconstruction of nucleus-associated microtubules in the division plane of vacuolated plant cells

The way in which transvacuolar strands radiating from the cell nucleus reorganize to form the phragmosome, within which division occurs, has been thoroughly studied in epidermal explants of Nautilocalyx lynchii. In recent years it has been established that the movement of the nucleus into the centre of large vacuolated cells such as these, in preparation for division, involves actin filaments. In the present study, the appearance and gradual reorganization of nucleus-associated microtubules (NAMTs) over the premitotic period is described. Epidermal explants fluorescently labelled with anti-tubulin were optically sectioned by confocal scanning laser microscopy, the sections reconstructed by an image processing computer and projected as rotating stereo pairs. This revealed that the NAMTs are a major component of the phragmosome, and that they change from a radiating to a planar distribution concomitantly with the ‘bunching’ of cortical MTs to form the pre-prophase band. The continuity of the two sets of MTs indicates that the band contains newly polymerized microtubules. Other recent studies on the division of vacuolated cells are reviewed and factors affecting the alignment of the division plane are discussed.     

Fluorescence in the tandem scanning microscope

Our studies have shown that the fluorescence mode can be used to good effect in both tandem scanning microscopes (TSM: direct view confocal microscopes) as well as confocal scanning laser microscopes (CSLM). Applications are presented which show that the two great advantages of TSM are real-time viewing and real colour, which allow faster use and interpretation. CSLM are complementary, not competitive, being currently more sophisticated for low-level fluorescence work. This is equally possible with available TSM, but requires further development using CCD cameras and image-processing systems.     

Second-order stereology for pores in translucent alumina studied by confocal scanning laser microscopy

The three-dimensional (3-D) arrangement of pores in translucent alumina was investigated with a confocal scanning laser microscope (CSLM). By moving the focal plane of the CSLM down into the material, a stack of serial thin optical sections was obtained to produce a 3-D image of the pores. Computer-based image analysis was used to obtain the coordinates of the pore centroids. The distance distribution function G(r) and the second-order functions K(r), L(r), H(r) and g(r) were used to analyse the spatial point pattern of the pore centroids. Estimates of the preceding functions obtained from eight stacks of sections were compared with the corresponding functions for a 3-D stationary Poisson point process, which served as a reference model for complete spatial randomness. The analysis suggested that the pore centroids were arranged in an aggregated pattern within a range of about 10 μm.     

Applications of confocal microscopy in industrial solid materials: some examples and a first evaluation

We report here the first results of the application of confocal scanning laser microscopy (CSLM) for the study of the microstructure of solid industrial materials. Glass-fibre-reinforced composites, heterogeneous and conductive polymers, homogeneous as well as heterogeneous catalyst (precursor) specimens and soils were examined. We conclude that both the fluorescence and reflection modes of CSLM can yield valuable information. In particular, the optical sectioning capability of CSLM appears to be of great value as it enables one to access the 3-D organization of the specimen without the need for a difficult and time-consuming specimen preparation procedure. However, local obscuration may be an important factor in confocal image formation, limiting the penetration capabilities of the technique for industrial materials.     

Three-dimensional visualization methods for confocal microscopy

Three-dimensional images of microscopic objects can be obtained by confocal scanning laser microscopy (CSLM). The imaging process in a CSLM consists of sampling a specific volume in the object and storing the result in a three-dimensional memory array of a digital computer. Methods are needed to visualize these images. In this paper three methods are discussed, each suitable in a specific area of application. For purposes where realistic rendering of solid or semi-transparent objects is required, an algorithm based on simulation of a fluorescence process is most suitable. When speed is essential, as for interactive purposes, a simple procedure to generate anaglyphs can be used. Both methods have in common that they require no previous interpretation or analysis of the image. When the study of an object imaged by CSLM involves analysis in terms of a geometrical model, sophisticated graphics techniques can be used to display the results of the analysis.     

Measurement of geometrical parameters in cocontinuous polymer blends: 3D versus 2D image analysis

Formulae of stereology are used to estimate 3D geometrical parameters of cocontinuous structures measured from 2D micrographs of polymer blends. 3D images of symmetric and nonsymmetric polymer blends made of fluorescently labelled polystyrene and styrene‐ran‐acrylonitrile copolymer were obtained with laser scanning confocal microscopy. Geometrical parameters of the blend interface, specifically volume fraction, surface area per unit volume (S _V ) and average of local mean curvature were measured directly from the 3D images and compared to the values estimated from analysis of a number of 2D slices combined with stereological relations. When the total length of phase boundary considered in the analysis of the 2D slices (L_Tot ) was at least 6000 times bigger than the characteristic length of the microstructure (S^?1_V ), the standard deviation for all the parameters measured became negligible. However, considerable discrepancies between the average values computed from 3D and 2D images were observed for any value of L_Tot . The mean curvature distribution was also measured from both the 3D images and the 2D slices. The distribution was estimated from the 2D slices but with a width about 2.4 times that of the true value obtained from the 3D images.     

In vivo determination of fibril orientation in plant cell walls with polarization CSLM

Congo Red fluorescence is used to detect cellulose in the wall of plant cells. The orientation of the cellulose fibrils is determined by using polarized light for excitation. The absorption characteristics of Congo Red make this approach a method of choice for applications with any standard confocal scanning laser microscope (CSLM). The semiquantitative character of CSLM observations combined with the non-toxicity of the stain allow a very fast and reliable assessment of cellulose orientation in the wall of living plant cells.     

Preliminary confocal scanning laser microscopy study of fluid inclusions in quartz

The initial results of the first dedicated confocal scanning laser microscopy (CSLM) study of fluid inclusions in quartz are presented. CSLM imaging of a large inclusion shows the quartz crystal to contain numerous small (< 1 μm), highly reflective inclusions arranged along planes in at least two directions that are not readily visible in transmitted light. The technique allows measurements to be made of the angular intersection and orientation of the planes in both two and three dimensions. Results suggest that larger inclusions (> 10 μm) occur where two planes of small inclusions intersect, and that the shape of the large inclusions is controlled by the angular relationship between intersecting planes.     

Working with the confocal scanning UV-laser microscope: specific DNA localization at high sensitivity and multiple-parameter fluorescence

The use of fast-staining DNA-specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488, 514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals. Here we report the characteristics and application of a CSLM, which was adapted for UV-excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple-parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double-stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes.     

A structural study of the bovine vaginal fluid at estrus.

The present study describes the structural components of the bovine vaginal fluid at estrus by scanning electron microscopy (SEM) following critical point- and freeze-drying preparation procedures. Confocal scanning laser microscopy (CLSM) was also used to evaluate the structural integrity of samples, and a control sample was assessed by adding sperm to the vaginal fluid. Samples were collected from 10 cows at the time of artificial insemination, prepared for SEM by using critical point- and freeze-drying procedures, gold coated, and observed by SEM. Mesh size and filament thickness were measured with an image analyzer. Of the 10 samples processed, 4 were considered altered following critical point drying. Compaction and lack of filaments were observed in these samples. A small area of one sample showed a honey comb-like structure when freeze drying was used. Nonoriented filaments with different thicknesses and with a network-like structure were observed throughout the remainder of the samples. Filaments throughout all samples were also observed by CSLM. After critical point drying, the mesh area ranged from 0.8 to 101.4 microns 2; the minor axis from 0.7 to 10.8 microns; and filament thickness from 40 to 442 nm. Using freeze drying, the mesh area ranged from 0.9 to 493.8 microns 2; the minor axis from 0.7 to 27.5 microns; and filament thickness from 40 to 800 nm. When samples were freeze dried, mesh values were similar to the interstrand channels observed by CSLM. In sperm-vaginal fluid samples, following critical point- or freeze-drying procedures, spermatozoa were oriented randomly in the vaginal fluid and did not seem to alter filamentous structure. Our data suggest that the freeze-drying procedure better preserves the true structural dimensions of the vaginal fluid. Furthermore, the filamentous structure of the vaginal fluid does not appear to impede sperm transport.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

Ultrastructural investigation of neurons identified and localized using the confocal scanning laser microscope

Studies of labeled neurons at the light-microscopic level often pinpoint a substructure of particular interest, i.e., a synapse or a spine. An ultrastructural investigation would explain a lot about how these structures arose, how they function, and how they are regulated. Finding a small region in a large block can require constant checking during sectioning, until past the structure. In our pursuit of the synaptic structure of varicosities on the axons of neurons identified physiologically and morphologically at the light level, we have combined confocal scanning laser microscopy (CSLM) with conventional and high-voltage electron microscopy (EM). CSLM images were collected in the reflection mode to view neurons filled with horseradish peroxidase and stained with nickel-intensified diaminobenzidine, which is compatible with EM. The CSLM optical sections provided a record of what one should expect to see at regular intervals throughout the depth of the tissue block. We have shown that the CSLM greatly simplified the task of localizing small structures in brain tissue prepared for EM.     

Optical sectioning with a fluorescence confocal SLM: procedures for determination of the 2-D digital modulation transfer function and for 3-D reconstruction by tessellation

We have determined the operational parameters of a confocal scanning laser microscope (CSLM) used for fluorescence imaging. The system performance was characterized by the modulation transfer function (MTF), calculated from an edge response function (ERF) corresponding to a standard test pattern overlaying fluorescence solutions on a microscopic slide. Signal truncation error was avoided by making the ERF continuous at its limits through superposition of a suitable linear curve. We determined an appropriate scanning step density by defining a compromise between the requirements of the sampling theorem with respect to aliasing and the need for minimal suppression of higher spatial frequencies. These procedures for choosing the sampling density of the total digital CSLM permit a systematic optimization of the image acquisition parameters. A reproducible digital image was obtained, an important prerequisite for subsequent 3-D image reconstruction. The latter was accomplished by first developing and applying a maximally automated algorithm for finding closed and distinct contours (corresponding to objects in the 2-D optical sections). The missing information between contour loops was then interpolated by tessellation (triangulation) using a minimal polygon edge length criterion capable of describing closed surfaces even for adjacent contours with highly dissimilar geometries. In this procedure, we define an average shift length which compensates for the inherent disadvantage of run length encoding algorithms applied to different starting points in successive planes. The surface segments were used to calculate 3-D representations by applying a shading model, in the present applications specifically to chromosome I and the nucleolus of polytenized Chironomus thummi salivary gland nuclei.