DNA-fluorescence of mammalian intra-nucleolar chromatin detected by confocal laser scanning microscopy (CLSM)

The complex spatial DNA distribution in the mammalian interphase nucleus was investigated in Feulgen stained thick sections through mouse trophoblast giant nuclei after Lowicryl embedding. DNA-fluorescence was visualized using confocal laser scanning microscopy. Our results show that the spatial arrangement of major interphase chromatin areas can be precisely documented, including the distribution of small intra-nucleolar chromatin zones.     

Characterization of theGeosiphon pyriforme symbiosome by affinity techniques: confocal laser scanning microscopy (CLSM) and electron microscopy

Summary Geosiphon pyriforme represents a photoautotrophic endosymbiosis of aGlomus-like fungus with the cyanobacteriumNostoc punctiforme. The fungus forms unicellular bladders of up to 2 mm in length and 0.5 mm in diameter growing on the soil surface and harboring the endosymbioticNostoc filaments. The cyanobacteria are located in a compartment (the symbiosome) bordered by a host membrane. The space between this symbiosome membrane (SM) and theNostoc cell wall is filled with an about 30–40 nm thick layer of amorphous material, which is present also in the regions of the symbiosome where noNostoc filaments are located. At these sites the amorphous material consists of a 20–30 nm thick layer separating the SM. The region between the SM and the cyanobacterium is defined as symbiosome space (SS). Fungal bladders, hyphae and free livingNostoc were analyzed by affinity techniques as well as the material occurring in the SS. FITC-coupled lectins with sugar specificity to -D-mannosyl/-D-glucosyl (Con A), N-acetyl--D-glucosamine oligomers (WGA), -L-fucosyl (UEA-I), -D-galactosyl (RCA-120), -D-galactosyl (BS-I-B₄), N-acetyl--D-galactosamine (HPA), and sialic acid (EBL) residues were tested. WGA binding and calcofluor white staining demonstrated that the bladder wall as well as the SS contain fibrillar chitin. Of the other lectins only Con A clearly labeled the symbiosome. On the contrary, the lectin binding properties of the slime produced by free livingNostoc-colonies indicate the presence of mannose, fucose, GalNAc, sialic acid, and galactose, while chitin or GlucNAc-oligomers could not be detected. The symbiosome was also investigated electron microscopically. WGA-gold binding confirmed the presence of chitin, while a slight PATAg reaction indicated some polysaccharidic molecules within the SS. Our results show that the amorphous material within the SS contains molecules typical of the fungal cell wall and suggest that the SM is related to the fungal plasma membrane. The applied lectins all bind to the hyphal surface, indicating a high molecular complexity. Mannosyl, -galactosyl, and sialic acid residues are strongly exposed at the outer cell wall layer, whereas GlucNAc, GalNAc, and -galactosyl residues seem to be present in smaller amounts. The symbiotic interface established between the fungus andNostoc inGeosiphon shows many similarities to that occurring between fungi and root cells in arbuscular mycorrhizas.Abbreviations AM arbuscular mycorrhiza - BS-I-B₄ Bandeiraea simplicifolia lectin I isolectin B₄ - CLSM confocal laser scanning microscopy - Con A Concanavalin A - EBL elderberry bark lectin I - FITC fluorescein isothiocyanate - HPA Helix pomatia agglutinin - PATAg periodic acid-thiocarbohydrazide-Ag proteinate - SM symbiosome membrane - SS symbiosome space - RCA-120 Ricinus communis agglutinin 120 - UEA-I Ulex europaeus agglutinin I - WGA wheat germ agglutinin Dedicated to Professor Dr. Peter Sitte at the occasion of his 65th birthday     

The musculature of three species of gastrotrichs surveyed with confocal laser scanning microscopy (CLSM)

The muscular system of gastrotrichs consists of circular, longitudinal and helicoidal bands that when analysed with confocal laser scanning microscopy, provide new insights into their functional organization and phylogenetic importance. We therefore undertook a comparative study of the muscle organization in three species of Gastrotricha from the orders Macrodasyida (Paradasys sp., Lepidodasyidae; Turbanella sp., Turbanellidae) and Chaetonotida (Polymerurus nodicaudus, Chaetonotidae). The general muscle organization of the marine interstitial macrodasyidans, Paradasys and Turbanella, not only confirms earlier observation on other species but also adds new details concerning the organization and number of helicoidal, longitudinal and other muscle bands (e.g. semicircular band). The freshwater, epibenthic–epiphytic chaetonotid, Polymerurus nodicaudus, has a similar muscular organization to other species of Chaetonotidae, especially species of Chaetonotus, Halichaetonotus and Lepidodermella. Perhaps unique to Polymerurus is the combined presence of an unbranched Rückenhautmuskel (also in Halichaetonotus and Lepidodermella) and a specialized dorsoventral caudal muscle, which flank the splanchnic component of the longitudinal muscles (only in Chaetonotus and Lepidodermella). This combination, together with the presence of splanchnic dorsoventral muscles, known only in Xenotrichulidae, implies a unique phylogenetic position for Polymerurus, and indicates a potential basal position of this taxon among the Chaetonotidae studied so far (i.e. Aspidiophorus, Chaetonotus, Halichaetonotus and Lepidodermella).     

The fluorescent probe HPTS as a phloem-mobile, symplastic tracer: an evaluation using confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) has been used to evaluatethe use of the fluorescent probe HPTS (8-hydroxypyrene-1,3,6-trisulphonicacid) as a symplastic tracer. HPTS-acetate was used to loadHPTS into the phloem and its movement was followed in threesystems where symplastic unloading has been proposed. In Arabidopsisroot tips and Abutilon nectaries the intercellular distributionof HPTS differed markedly from that observed with 5-(and 6)-carboxyfluorescein(CF)- HPTS was observed in the nuclei and cytoplasm whilst CFwas rapidly transferred into the vacuoles. In contrast, bothHPTS and CF accumulated in the vacuoles of the vascular parenchymaand nucellus cells following unloading from the phloem of thedeveloping barley caryopsis. The results indicate that HPTShas a number of advantages as a symplastic probe compared withCF. The findings are discussed in relation to the influenceof vacuolar sequestration on dye distribution. Key words: Confocal laser scanning microscopy (CLSM), HPTS, intercellular transport, phloem (unloading)     

Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM).

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.     

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     

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.     

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.Abbreviations BSA Bovine serum albumin - CGRP calcitonin generelated peptide - DiA carbocyanine dye A - DiI carbocyanine dye I - dmnX dorsal motor nucleus of vagus - DMSO dimethylsulfoxide - ENK enkephalin - FITC fluorescin isothiocyanate - NADPH diaphorase nicotinamide adenine diphosphate - NPY neuropeptide Y - NTS nucleus tractus solitarii - PBS phosphate-buffered saline - VIP vasoactive intestinal peptide - WGA-HRP wheat-germ agglutinine-horseradish peroxidase     

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.     

Cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM) in the study of neutral lipid dynamics in Paramecium primaurelia mating types during cell line development

BACKGROUND: In Paramecium primaurelia, an exconjugant cell can produce two lines with different mating capacities. Mating type II cells can form a higher food vacuole number and digest the nutrient taken up in a shorter time; thus, mating type II cells grow at a faster rate than do mating type I cells. The present study was done to determine whether cells that ingest more nutrients also have a larger amount of storage lipids. METHODS: Quantitative and qualitative determinations of neutral lipids were obtained by means of cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM), respectively, by using nile red on cells in different physiologic states. RESULTS: Lipid droplet number and neutral lipid content were higher in mating type II cells than in mating type I cells in the early logarithmic growth phase (i.e., immature well-fed cells). These values were reversed during the middle and the late logarithmic phases and became equal in the stationary phase (i.e., mature starved cells). In well-fed cells maintained with food excess, differences in neutral lipid content between the two mating types also were present in mature cells. CONCLUSIONS: Although differences between mating type I and mating type II lines were not correlated to cell size, a relation was found between lipid content and food ingestion capacity. A depletion of bacteria in the culture medium could be responsible for the lack of differences in mature starved cells. CLSM allowed us to gather volume information about the lipid droplet distribution within the cell.     

Three-dimensional visualization of Salmonella attachment to poultry skin using confocal scanning laser microscopy

K.Y. KIM, J.F. FRANK AND S.E. CRAVEN. 1996. The objective of this study was to locate the position of attached or entrapped Salmonella cells in poultry skin. Confocal scanning laser microscopy (CSLM) was used to obtain optical sections of intact poultry skin without artefacts associated with dehydration and other sample preparation techniques. A technique was developed to prevent compression of the poultry skin during CSLM operation. Images of bacteria and poultry skin were obtained after staining with Pyronin-Y. Data indicated that Salmonella cells were mostly located in the cervices and feather follicles. Salmonella in feather follicle floated freely in surrounding liquid even after the skin was thoroughly rinsed.     

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.     

Neutral red as a probe for confocal laser scanning microscopy studies of plant roots

BACKGROUND AND AIMS: Neutral red (NR), a lipophilic phenazine dye, has been widely used in various biological systems as a vital stain for bright-field microscopy. In its unprotonated form it penetrates the plasma membrane and tonoplast of viable plant cells, then due to protonation it becomes trapped in acidic compartments. The possible applications of NR for confocal laser scanning microscopy (CLSM) studies were examined in various aspects of plant root biology. METHODS: NR was used as a fluorochrome for living roots of Phaseolus vulgaris, Allium cepa, A. porrum and Arabidopsis thaliana (wild-type and transgenic GFP-carrying lines). The tissues were visualized using CLSM. The effect of NR on the integrity of the cytoskeleton and the growth rate of arabidopsis primary roots was analysed to judge potential toxic effects of the dye. KEY RESULTS: The main advantages of the use of NR are related to the fact that NR rapidly penetrates root tissues, has affinity to suberin and lignin, and accumulates in the vacuoles. It is shown that NR is a suitable probe for visualization of proto- and metaxylem elements, Casparian bands in the endodermis, and vacuoles in cells of living roots. The actin cytoskeleton and the microtubule system of the cells, as well as the dynamics of root growth, remain unchanged after short-term application of NR, indicating a relatively low toxicity of this chemical. It was also found that NR is a useful probe for the observation of the internal structures of root nodules and of fungal hyphae in vesicular-arbuscular mycorrhizas. CONCLUSIONS: Ease, low cost and absence of tissue processing make NR a useful probe for structural, developmental and vacuole-biogenetic studies of plant roots with CLSM.     

Clearing pigmented insect cuticle to investigate small insects' organs in situ using confocal laser-scanning microscopy (CLSM)

Various microscopic techniques allow investigating structures from submicron to millimeter range, however, this is only possible if the structures of interest are not covered by pigmented cuticle. Here, we present a protocol that combines clearing of pigmented cuticle while preserving both, hard and soft tissues. The resulting transparent cuticle allows confocal laser-scanning microscopy (CLSM), which yields high-resolution images of e.g. the brain, glands, muscles and fine cuticular structures. Using a fluorescent dye, even single labeled neurons can be visualized and resolved up to an imaging depth of 150 μm through the cleared cuticle. Hydrogen-peroxide, which was used to clear the cuticle, does not preclude immunocytochemical techniques, shown by successful labeling of serotonin-immunoreactive neurons (5HT-ir) in the ants' brain. The ‘transparent insect protocol’ presented here is especially suited for small arthropods where dissection of organs is very demanding and difficult to achieve. Furthermore, the insect organs are preserved in situ thus allowing a more precise three-dimensional reconstruction of the structures of interest compared to, e.g., dissected or sectioned tissue.     

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.