CTC staining and counting of actively respiring bacteria in natural stone using confocal laser scanning microscopy

A method was established for staining and counting of actively respiring bacteria in natural stone by using the tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride (CTC) in combination with confocal laser scanning microscopy (CLSM). Applying 5 mM CTC for 2 h to pure cultures of representative stone-inhabiting microorganisms showed that chemoorganotrophic bacteria and fungi-in contrast to lithoautotrophic nitrifying bacteria-were able to reduce CTC to CTF, the red fluorescing formazan crystals of CTC. Optimal staining conditions for microorganisms in stone material were found to be 15 mM CTC applied for 24 h. The cells could be visualized on transparent and nontransparent mineral materials by means of CLSM. A semi-automated method was used to count the cells within the pore system of the stone. The percentage of CTC-stained bacteria was dependent on temperature and humidity of the material. At 28 degrees C and high humidity (maximum water holding capacity) in the laboratory, about 58% of the total bacterial microflora was active. On natural stone exposed for 9 years at an urban exposure site in Germany, 52-56% of the bacterial microflora was active at the east, west, and north side of the specimen, while only 18% cells were active at the south side. This is consistent with microclimatic differences on the south side which was more exposed to sunshine thus causing UV and water stress as well as higher temperatures on a microscale level. In combination with CLSM, staining by CTC can be used as a fast method for monitoring the metabolic activity of chemoorganotrophic bacteria in monuments, buildings of historic interest or any art objects of natural stone. Due to the small size of samples required, the damage to these objects and buildings can be minimized.     

Improved detection of in situ hybridization by laser scanning confocal microscopy.

Laser scanning confocal microscopy (LSCM) offers a significant improvement over conventional bright-field and dark-field light microscopy for producing images of silver grains in autoradiograms of specimens prepared by in situ hybridization. The out-of-focus image of the background silver grains present in the emulsion is eliminated from the in-focus image of the radioactive probe associated with the cells by optical sectioning with the LSCM operated in a reflected light mode. The improved images produced by the LSCM provide a significant increase in the sensitivity of detecting positively labeled cells and tissues prepared by in situ hybridization. The power of this detection method is demonstrated using samples of HIV-infected human peripheral blood cells, tissue sections of human placenta and human skin. It is anticipated that the method can be universally applied to samples prepared by in situ hybridization techniques.     

Rotifer muscles as revealed by phalloidin-TRITC staining and confocal scanning laser microscopy

By combining phalloidin‐TRITC staining with confocal scanning laser microscopy (CSLM), the pattern of the musculature in two species of Rotifera, Euchlanis dilatata unisetata and Brachionus quadridentatus is revealed. The same general muscle pattern prevails in both species. The major components of the body wall musculature are: 1. retractor muscles (5 pairs in E. dilatata unisetata and 3 pairs in B. quadridentatus); 2. Two pairs of dorso‐ventral muscles; 3. Two pairs of perpendicular muscles (in E. dilatata unisetata); 4. retractors of the corona (median, lateral and ventral); 5. Foot retractors. In addition, three pairs of cutaneo‐visceral muscles and visceral muscles (including mastax muscles) are described. The sphincter of the corona was found only in B. quadridentatus. The high degree of muscle differentiation points to a high level of development of rotifer muscular system.     

Osmotic water permeability measurements using confocal laser scanning microscopy

 We have developed a method for measurement of plasma membrane water permeability (P _f) in intact cells using laser scanning confocal microscopy. The method is based on confocal recording of the fluorescence intensity emitted by calcein-loaded adherent cells during osmotic shock. P _f is calculated as a function of the time constant in the fluorescence intensity change, the cell surface-to-volume ratio and the fractional content of the osmotically active cell volume. The method has been applied to the measurement of water permeability in MDCK cells. The cells behaved as linear osmometers in the interval from 100 to 350 mosM. About 57% of the total cell volume was found to be osmotically inactive. Water movement across the plasma membrane in intact MDCK cells was highly temperature dependent. HgCl₂ had no effect on water permeability, while amphotericin B and DMSO significantly increased P _f values. The water permeability in MDCK cells transfected with aquaporin 2 was an order of magnitude higher than in the intact MDCK cell line. The water permeability of the nuclear membrane in both cell lines was found to be unlimited. Thus the intranuclear fluid belongs to the osmotically active portion of the cell. We conclude that the use of confocal microscopy provides a sensitive and reproducible method for measurement of water permeability in different types of adherent cells and potentially for coverslip-attached tissue preparations. Received: 12 June 1999 / Revised version: 21 February 2000 / Accepted: 25 February 2000     

Localization of cerium-based reaction products by scanning laser reflectance confocal microscopy

Scanning laser confocal microscopy was utilized to visualize sites of hydrogen peroxide release from stimulated neutrophils and lysosomal acid phosphatase in these and other cells using cerium in the detection systems. Imaging of the cerium-containing reactions was achieved by employing the reflectance mode of this instrument. Localization of these products at the light microscope level was direct and did not require other reactions to generate a visible product. This new approach to cerium cytochemistry should prove useful for many applications.     

Detection of diaminobenzidine reactions using scanning laser confocal reflectance microscopy

We used scanning laser confocal microscopy to visualize sites of peroxidatic activity as detected by the diaminobenzidine (DAB) reaction. Imaging was achieved by employing the reflectance mode of this instrument. Intense reflectance was detected after DAB localization of endogenous granule-associated myeloperoxidase in neutrophils and of the exogenous tracer horseradish peroxidase in mouse oocytes. Detection of DAB reaction products with confocal reflectance microscopy will probably be an important addition to the utility of this cytochemical technique.     

Worm morphology of Schistosoma japonicum using confocal laser scanning microscopy

Male and female Schistosoma japonicum worms have dissimilar appearances in their final host. In this study, a morphometric and morphological assessment of whole worms derived from unisexual and mixed infections in mice was conducted using confocal laser scanning microscopy. Worms from mixed infections showed significant morphological changes between 15 and 25 days post-infection (PI). On the fifteenth day PI, 33% of males had formed the conspicuous gynecophoric canal, but only 8% of them had testicular lobes containing a few germinative cells; 13% of females had incipient ovaries with a few immature ovarian cells inside. On the twentieth day PI, the testicular lobes contained more germinative cells in all male worms, while female worms presented vitelline glands. On the twenty-fifth day PI, more germinative cells were observed in the male testicular lobes, and differentiated cells were present in the female ovaries. All worms had fully developed reproductive organs from 30 days PI onwards. Morphometric analysis showed significant differences between mixed and unisexual infections at 35 days PI. Ovaries of worms from unisexual infections contained cells in one stage of maturation and vitelline glands had undifferentiated cells. Our study of S. japonicum provides a detailed comparison of different morphological traits from worms of mixed and unisexual infections throughout development.     

Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)     

Three-dimensional imaging of plant cuticle architecture using confocal scanning laser microscopy

Full appreciation of the roles of the plant cuticle in numerous aspects of physiology and development requires a comprehensive understanding of its biosynthesis and deposition; however, much is still not known about cuticle structure, trafficking and assembly. To date, assessment of cuticle organization has been dominated by 2D imaging, using histochemical stains in conjunction with light and fluorescence microscopy. This strategy, while providing valuable information, has limitations because it attempts to describe a complex 3D structure in 2D. An imaging technique that could accurately resolve 3D architecture would provide valuable additions to the growing body of information on cuticle molecular biology and biochemistry. We present a novel application of 3D confocal scanning laser microscopy for visualizing the architecture, deposition patterns and micro-structure of plant cuticles, using the fluorescent stain auramine O. We demonstrate the utility of this technique by contrasting the fruit cuticle of wild-type tomato ( Solanum lycopersicum cv. M82) with those of cutin-deficient mutants. We also introduce 3D cuticle modeling based on reconstruction of serial optical sections, and describe its use in identification of several previously unreported features of the tomato fruit cuticle.     

Visualising fouling of a chromatographic matrix using confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) was used to visualise the spatial location of foulants during the fouling of Q Sepharose FF matrix in finite batch experiments and for examining the subsequent effectiveness of clean-in-place (CIP) treatments in cleaning the heavily fouled beads. Beads were severely fouled with partially clarified E. coli homogenate by contacting the beads with the foulant for contact times of 5 min, 1 or 12 h. The use of two different fluorescent dyes, PicoGreen and Cy5.5, for labelling genomic PicoGreen-labelled dsDNA and protein respectively, allowed the direct observation of the chromatographic beads. The extent of fouling was assessed by measuring the subsequent adsorption of Cy5.5-labelled BSA to the beads. Control studies established that the labelling of BSA did not affect significantly the protein properties. In the control case of contacting the unfouled matrix with Cy5.5-labelled BSA, protein was able to penetrate the entire matrix volume. After fouling, Cy5.5-labelled BSA was unable to penetrate the bead but only to bind near the bead surface where it slowly displaced PicoGreen-conjugated dsDNA, which bound only at the exterior of the beads. Labelled host cell proteins bound throughout the bead interior but considerably less at the core; suggesting that other species might have occupied that space. The gross levels of fouling achieved drastically reduced the binding capacity and maximum Cy5.5-labelled BSA uptake rate. The capacity of the resin was reduced by 2.5-fold when incubated with foulant for up to 1 h. However, when the resin was fouled for a prolonged time of 12 h a further sixfold decrease in capacity was seen. The uptake rate of Cy5.5-labelled BSA decreased with increased fouling time of the resin. Incubating the fouled beads in 1 M NaCl dissociated PicoGreen-labelled dsDNA from the bead exterior within 15 min of incubation but proved ineffective in removing all the foulant protein. Cy5.5-labelled BSA was still unable to bind beyond the outer region of the beads. A harsher CIP treatment of 1 M NaCl dissolved in 1 M NaOH was also ineffective in removing all the foulant protein but did remove PicoGreen-conjugated dsDNA within 15 min of incubation. Cy5.5-labelled BSA was able to bind throughout the bead interior after this more aggressive CIP treatment but at a lower capacity than in the case of fresh beads. The competitive adsorption of BacLight Red-labelled whole cells or cell debris and PicoGreen-conjugated dsDNA was also visualised using CSLM.     

Fluorescent in situ hybridisation on tissue sections: a quantitative approach with confocal laser scanning microscopy

The use of fluorescent detection methods in association with digital microscopy technologies is an innovative approach for tissue localisation of messenger RNA. The success of such methods relies on the tissue preservation, local availability of the probe and on the existence of high resolution tridimensional analysis systems. Cryostatic sections, mild denaturation, short oligonucleotide probes (20mer) and confocal laser scanning microscopy allow the fulfillment of all these conditions avoiding photobleaching and tissue autofluorescence. In this paper, we describe in detail a method for in situ hybridisation set up with digoxigenin-coupled oligonucleotide complementary to beta-actin mRNA as a probe and an anti-hapten fluorescent antibody as second step for detecting specific hybridisation. Fluorescence was analysed by means of a confocal laser scanning microscope (CLSM) that provides images with low out-of-focus blurring also with relatively low numerical aperture (NA) objectives. We propose also an easy method to perform semi-quantitative thresholding analysis which allows to discriminate between background and specific signal.     

Principles and practices of laser scanning confocal microscopy

The laser scanning confocal microscope (LSCM) is an essential tool for many biomedical imaging applications at the level of the light microscope. The basic principles of confocal microscopy and the evolution of the LSCM into today's sophisticated instruments are outlined. The major imaging modes of the LSCM are introduced including single optical sections, multiple wavelength images, three-dimensional reconstructions, and living cell and tissue sequences. Practical aspects of specimen preparation, image collection, and image presentation are included along with a primer on troubleshooting the LSCM for the novice.     

Power and limits of laser scanning confocal microscopy

In confocal microscopy, the object is illuminated and observed so as to rid the resulting image of the light from out-of-focus planes. Imaging may be performed in the reflective or in the fluorescence mode. Confocal microscopy allows accurate and nondestructive optical sectioning in a plane perpendicular or parallel to the optical axis of the microscope. Further digital three-dimensional treatments of the data may be performed so as to visualize the specimen from a variety of angles. Several examples illustrating each of these possibilities are given. Three-dimensional reconstitution of nuclear components using a cubic representation and a ray-tracing based method are also given. Instrumental and experimental factors can introduce some bias into the acquisition of the 3-D data set: self-shadowing effects of thick specimens, spherical aberrations due to the sub-optimum use of the objective lenses and photobleaching processes. This last phenomenon is the one that most heavily hampers the quantitative analysis needed for 3-D reconstruction. We delineate each of these problems and indicate to what extent they can be solved. Some tips are given for the practice of confocal microscope and image recovery: how to determine empirically the thickness of the optical slices, how to deal with extreme contrasts in an image, how to prevent artificial flattening of the specimens. Finally, future prospects in the field are outlined. Particular mention of the use of pulsed lasers is made as they may be an alternative to UV-lasers and a possible means to attenuate photodamage to biological specimens.     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.     

Detection of submicroscopic magnetite particles using reflectance mode confocal laser scanning microscopy

Light microscopy and transmission electron microscopy work at such different scales that some components of cells may be too small to detect using light microscopy but too dispersed among cells within tissues to be discovered using electron microscopy. We have used reflectance mode confocal laser scanning microscopy to detect single-domain magnetite crystals in both live and resin-embedded preparations of magnetotactic bacteria. We show that reflections from bacterial cells are uniquely associated with the magnetite, which underpins the magnetotactic response of the bacteria. En bloc viewing shows that relatively large volumes of material can be searched with sufficient resolution to enable detection of submicroscopic particles. The techniques reported here may be of interest to others wishing to detect submicroscopic objects dispersed in large volumes of tissue.     

Noninvasive in situ observation of the crystallization kinetics of biological macromolecules by confocal laser scanning microscopy

High-resolution confocal laser scanning microscopy (CLSM) is a powerful tool for in situ observation and analysis of protein crystal growth kinetics. Because the resolution of CLSM is not diffraction-limited by the object, it is possible to visualize, under certain conditions, objects in molecular dimensions. A modified batch technique is applied which allows the growth kinetics of sufficiently small crystallites fixed at the lower side of a cover glass, within a hanging drop, to be studied in reflected light near the total reflection angle. A gap, or cavity, filled with solution is formed between the cover glass and the upper crystal face, which acts to fix small crystallites by hydrodynamic friction forces. The cavity height enables the propagation of molecular steps across the upper crystal face without constraint, so that the propagation velocity and geometrical parameters can be measured by CLSM. The layer growth kinetics of monoclinic crystallites of a long-acting insulin derivative (Insulin Glargine) is investigated. For a twofold supersaturation of the solution, the growth is governed by 2D nucleation at the edges of the crystallites followed by a spreading of molecular steps. The layer growth kinetics are well fitted by the simple cubic kinetic lattice model. We find that only about one of a thousand solute (protein) molecules which push a kink place due to their Brownian motion becomes really incorporated into the growing crystal.     

Use of UV excitation in confocal laser scanning fluorescence microscopy

By making only minor modifications, we adapted a conventional confocal beam-scanning laser microscope for the recording of UV-excited fluorescence. The major, and most expensive, change is that we coupled an external UV argon ion laser, providing the wavelengths 334, 351 and 364 nm, to the microscope scanner. We also replaced some optical components to obtain improved transmission and reflection properties in the UV. Only easily obtainable and inexpensive off-the-shelf components were used. The most serious problem encountered was the chromatic aberration of the microscope objective when using both UV and visible wavelengths. This is of no consequence in conventional microscopy where good imaging properties are important only in the visible region. In confocal microscopy on the other hand, good imaging properties are necessary for both the exciting and fluorescent light. Rather than having new optics designed, we tried with simple means to reduce the effects of the chromatic aberration to a tolerable level. This was done by mechanical adjustments in the ray-path. In addition we also tested two mirror objectives, which are inherently free from chromatic aberrations. However, such objectives have rather limited numerical apertures and are not of the immersion type. Their value in biomedical applications is therefore limited.The objective most frequently used in our experiments was a 63/1.25 oil-immersion fluorite. Without any compensation this objective had a depth resolution in UV-excited confocal fluorescence that was an order of magnitude worse than when using visible-light excitation. The useful field of view was also very small due to lateral chromatic aberration. By simple means we managed to improve the depth resolution by a factor of 4.4, and at the same time increase the useful field of view substantially. Still, the depth resolution was worse than what is obtained using visible light excitation. We think this is due to the fact that after compensation the objective is working with an incorrect tube length.Using the modified instrument, we recorded specimens labelled with AMCA and Fluoro-Gold, obtaining 1.5 μm thick optical sections.     

Visualization of alginate-poly-L-lysine-alginate microcapsules by confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) was used to study the distribution of polymers and cross-linking ions in alginate-poly-L-lysine (PLL) -alginate microcapsules made by fluorescent-labeled polymers. CLSM studies of Ca-alginate gel beads made in the presence and absence of non-gelling sodium ions revealed a more inhomogeneous distribution of alginate in beads formed in the absence of non-gelling ions. In the formation of alginate-PLL capsules, the polymer gradients in the preformed gel core were destabilized by the presence of non-gelling ions in the washing step and in the PLL solution. Ca-alginate gels preserved the inhomogeneous structure by exposure to ion-free solution in contrast to exposure to non-gelling ions (Na(+)). By exchanging Ca(2+) with Ba(2+) (10 mM), extremely inhomogeneous gel beads were formed that preserved their structure during the washing and exposure to PLL in saline. PLL was shown to bind at the very surface of the alginate core, forming a shell-like membrane. The thickness of the PLL-layer increased about 100% after 2 weeks of storage, but no further increase was seen after 2 years of storage. The coating alginate was shown to overlap the PLL layer. No difference in binding could be observed among coating alginates of different composition. This paper shows an easy and novel method to study the distribution of alginate and PLL in intact microcapsules. As the labeling procedures are easy to perform, the method can also be used for a variety of other polymers in other microencapsulation systems.