Imaging modes in confocal scanning light microscopy (CSLM)

The optical arrangement for confocal scanning light microscopy can be incorporated in various imaging modes. Light microscopical specimens can be imaged with contrast enhanced, under γ-control, inverted, etc. In interference, conditions can be set such that either pure phase or pure amplitude images result. Stereoscopic images at arbitrary aspect ratios can be realized in CSLM by electronic processing of the data obtained when the specimen is sampled with more than one confocal point concurrently. Also forms of differential imaging either amplitude or phase are possible. The coupling of these imaging modes with the improved resolving powers of CSLM results in some unique imaging opportunities, especially of value for high resolution light microscopy of living specimens.     

An optical spectrometer for a confocal scanning laser microscope

An optical spectrometer for the visible range has been developed for the confocal scanning laser microscope (CSLM) Phoibos 1000. The spectrometer records information from a single point or a user-defined region within the microscope specimen. A prism disperses the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit cools the diode array, thereby reducing the detector dark current to a level, which allows integration times of up to 60 s. The spectral resolving power, λ/Δλ, ranges from 400 at λ = 375 nm to 100 at λ = 700 nm. Since the entrance aperture of the spectrometer has the same diameter as the detector aperture of the CSLM, the three-dimensional spatial resolution for spectrometer readings is equivalent to that of conventional confocal scanning, that is, down to 0.2 μm lateral and 0.8 μm axial resolution with an N.A.=1.3 objective.     

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.     

Examination of microhardness indentation-induced subsurface damage in alumina platelet reinforced borosilicate glass using confocal scanning laser microscopy

Borosilicate glass and borosilicate glass matrix composites reinforced with 10, 15 and 30 vol.% alumina platelets have been indented and the subsurface lateral cracking examined by confocal scanning laser microscopy (CSLM). The suitability of the CSLM technique as a quick and nondestructive method of obtaining three-dimensional information of subsurface damage in dispersion reinforced brittle matrix composite materials was demonstrated. The addition of alumina platelets to the glass matrix has resulted in a reduction in the extent and depth of subsurface damage due to indentation, and hence may make the material more resistant to erosive wear. This damage development may be a consequence of the presence of residual compressive stresses in the matrix and the strong platelet/matrix interfacial bonding.     

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 simple method for overcoming some problems when observing thick reflective biological samples with a confocal scanning laser microscope

A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological samples with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslips (‘sandwich’ style) and the optical tomography was performed from both sides by turning the ‘sandwich’ upside-down. The samples were impregnated using standard Golgi–Cox, ‘rapid Golgi’ or other silver methods. The ability to turn the ‘sandwich’ upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e. near the lower surface of the specimen, or when it is opaque to the laser beam or excessively reflective.     

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.     

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.     

Confocal scanning light microscopy with high aperture immersion lenses

The imaging characteristics of a confocal scanning light microscope (CSLM) with high aperture, immersion type, lenses (N.A. = 1·3) are investigated. In the confocal arrangement the images of the illumination and detector pinholes are made to coincide in a common point, through which the object is scanned mechanically. Results show that for point objects the theoretically expected improved response by a factor of 1·4 in comparison with standard microscopy can indeed be realized. Low side lobe intensity and absence of glare permits the imaging at high resolution of weak details close to strong features. A further improvement by a factor of 1·25 in point resolution in CSLM is found after apodization with an annular aperture. Due to the scanning approach all possibilities of electronic image processing become available in light microscopy.     

Application of confocal scanning laser microscopy in experimental pathology

Confocal scanning laser microscopy (CSLM) represents an exciting new tool for scientific disciplines which focus on mechanistic studies such as experimental pathology. Enhanced resolution in the specimen plane and rejection of out-of-focus fluorescence flare allow analysis of specific nucleic acid sequences, enzymes, structural macromolecules, and cellular homeostasis utilizing fluorescent probes. Four different experimental applications are discussed which utilize CSLM to evaluate pathological processes at the subcellular, cellular, and tissue levels. Programmed cell death, or apoptosis, is a natural process of significance both during development and as a response to toxic stimuli. CSLM-imaging of nuclei of human B lymphoblastoid cells following exposure to a monofunctional alkylating agent suggests that the degradation of chromatin characteristic of apoptosis may occur in asymmetric patterns. Surfactant apoprotein-A is the major non-serum protein component of pulmonary surfactant and is essential for the extracellular function of surfactant. CSLM of alveolar type II cells suggests that apoprotein-A is present in both the cytoplasm, predominantly in lamellar bodies, and in the nucleus. The tumor promoter, phorbol myristate acetate, rapidly stimulated the formation of vacuoles in human neutrophils. CSLM using Lucifer Yellow as a probe suggests that cylindrical vacuoles are formed by fluid-phase pinocytosis. The blood-nerve barrier (BNB) in peripheral nerves may be an important target during toxin-induced neuropathies. Ricin-induced permeability of the BNB in the rat was rapidly visualized by CSLM as leakage of fluorescein isothiocynate (FITC)-dextran into the endoneurial compartment.     

Fluorescence photobleaching recovery in the confocal scanning light microscope

Measurement of mobilities of species in liquid systems is of great importance for understanding a number of dynamic phenomena. A well known method for measuring mobilities driven by diffusion is fluorescence photobleaching recovery (FPR), also known as fluorescence recovery after photobleaching (FRAP). New FPR recovery equations for three-dimensional (3-D) apertured scanning using a Gaussian approximation for the axial beam profile have been successfully developed and found to provide a solid basis for extraction of the lateral diffusion coefficient from confocal scanning light microscopy (CSLM)-FPR experimental data. The 2-D diffusion coefficients of fluorescent species can be successfully measured by FPR in the CSLM, which has the great advantage that bleaching can be targeted at a well-defined volume element in bulk samples. Two-dimensional diffusion coefficients of 45-nm latex spheres, of FITC molecules and of a 2·45-nm protein-FITC complex in water-glycerol mixtures, measured by FPR in the CSLM, are in close agreement with those calculated from the size of the diffusing species and viscosity of the medium. Diffusion coefficients as high as 2 times 10^?6 cm²/s can be measured.     

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.     

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.     

Microfractography of granitic rocks under confocal scanning laser microscopy

Scanning laser microscopy, in the confocal mode (CSLM) has been applied to a granitic rock to characterize its fissure space. The technique provides a unique three-dimensional picture of the rock microfractography. CSLM is unique in observing fine details of the fractographic network (connectivity, tortuosity, etc.), its geometry and its relation to other rock-forming components. The fractographic images with standard fluorescence microscopy are compared with those obtained with CSLM. The examples presented emphasize the advantages of CSLM: three-dimensional visualization of the microfractographic network, crack connectivity, automatic evaluation of direction and slope of fissures. These studies are related to the migration of radionuclides in the geosphere. The relations between potentially water-conducting open fissures, and the rock-forming minerals provide a means of modelling the ‘radionuclide retardation mechanism’, a security factor in their definitive storage in rock masses.     

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.     

An FFT-based method for attenuation correction in fluorescence confocal microscopy

A problem in three-dimensional imaging using a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. A new method is proposed to correct for these effects. The approach, valid for weak attenuation, consists of multiplying the measured fluorescence intensity by a correction factor involving a convolution integral of the measured signal, which can be computed efficiently by the fast Fourier transform. Analytical and numerical estimates are given for the degree of attenuation under which the method is valid, and the method is applied to various test images. A real CSLM image is restored. Finally, the method is compared with a recent iterative method with regard to numerical accuracy and computational efficiency.     

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.     

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.     

Digital differential confocal microscopy based on spatial shift transformation

Differential confocal microscopy is a particularly powerful surface profilometry technique in industrial metrology due to its high axial sensitivity and insensitivity to noise. However, the practical implementation of the technique requires the accurate positioning of point detectors in three‐dimensions. We describe a simple alternative based on spatial transformation of a through‐focus series of images obtained from a homemade beam scanning confocal microscope. This digital differential confocal microscopy approach is described and compared with the traditional Differential confocal microscopy approach. The ease of use of the digital differential confocal microscopy system is illustrated by performing measurements on a 3D standard specimen.