Measurement differences between turbidity instruments, and their implications for suspended sediment concentration and load calculations: A sensor inter-comparison study.

The use of turbidity for indicating environmentally detrimental levels of suspended and colloidal matter in freshwater systems, and for defining acceptable water quality standards in national and European drinking water regulations, is well established. Turbidity is therefore frequently adopted as a surrogate for suspended sediment concentrations (SSC), or as a relative and objective measure of water clarity in monitoring programmes. Through systematic, controlled experimentation, we tested the response of 12 commercially available turbidity sensors, of various designs, to gauge their measurement consistency when benchmarked against pre-prepared sediment suspensions of known SSC. Results showed that despite calibration to a Formazin standard, sensor responses to identical SSC solutions (in the range of 20-1000 mg L-1) varied considerably. For a given SSC, up to five-fold differences in recorded turbidity were recorded across the tested instruments. Furthermore, inconsistent measurements were identified across instruments, regardless of whether they operated using backscatter or side-scatter optical principles. While the findings may have implications for compliance with turbidity-based water quality standards, they are less likely to be an issue when turbidity is being used as a surrogate for SSC, provided that instrument use remains constant and that instrument drift is not an issue. In this study, a field comparison of a subset of four study sensors showed that despite very different absolute turbidity readings for a given SSC, well correlated and reliable turbidity - SSC ratings were established (as evidenced by r2 coefficients from 0.92 to 0.98). This led to reasonably consistent suspended sediment load estimates of between 64.7 and 70.8 tonnes for a rainfall event analysed. This study highlights the potential for issues to arise when interpreting water turbidity datasets that are often assumed to be comparable, in that measurement inconsistency of the type reported here may remain unknown to water resource decision-makers and practitioners.

Morphology of astroglial cells is controlled by beta-adrenergic receptors.

Astroglial cells in vivo and in vitro respond to hormones, growth factors, and neurotransmitters by changing from an epithelial-like to stellate morphology. We have studied the temporal relationship between receptor activation, second messenger mobilization, and morphological changes using LRM55 astroglial cells. Maintenance of an altered morphology required continuous beta-adrenergic receptor activation. These changes appeared to be mediated by cAMP since they were elicited by its analogue, dibutyryl cAMP, and by forskolin, a direct activator of adenylate cyclase. Changes in cell morphology may require a relatively small increase in intracellular cAMP, since receptor-stimulated changes in cAMP levels were transient and peaked approximately 5 min after receptor activation while changes in morphology took at least 30 min to reach a new steady state. Time-lapse videomicroscopy and high voltage electron microscopy indicated that receptor activation resulted in a sequence of morphological events. Time-lapse observations revealed the development and enlargement of openings through the cytoplasm associated with cytoplasmic withdrawal to the perinuclear region and process formation. Higher resolution high voltage electron microscopy indicated that the transition to a stellate morphology was preceded by the appearance of two distinct cytoplasmic domains. One contained an open network of filaments and organelles. The other was characterized by short broad cytoplasmic filaments. The first domain was similar to cytoplasm in control cells while the second was associated with the development and enlargement of openings through the cytoplasm and regions of obvious cytoplasmic withdrawal.

Constant pressure fluid infusion into rat neocortex from implantable microfluidic devices.

Implantable electrode arrays capable of recording and stimulating neural activity with high spatial and temporal resolution will provide a foundation for future brain computer interface technology. Currently, their clinical impact has been curtailed by a general lack of functional stability, which can be attributed to the acute and chronic reactive tissue responses to devices implanted in the brain. Control of the tissue environment surrounding implanted devices through local drug delivery could significantly alter both the acute and chronic reactive responses, and thus enhance device stability. Here, we characterize pressure-mediated release of test compounds into rat cortex using an implantable microfluidic platform. A fixed volume of fluorescent cell marker cocktail was delivered using constant pressure infusion at reservoir backpressures of 0, 5 and 10 psi. Affected tissue volumes were imaged and analyzed using epifluorescence and confocal microscropies and quantitative image analysis techniques. The addressable tissue volume for the 5 and 10 psi infusions, defined by fluorescent staining with Hoescht 33342 dye, was significantly larger than the tissue volume addressed by simple diffusion (0 psi) and the tissue volume exhibiting insertion-related cell damage (stained by propidium iodide). The results demonstrate the potential for using constant pressure infusion to address relevant tissue volumes with appropriate pharmacologies to alleviate reactive biological responses around inserted neuroprosthetic devices.

Fluvial-controlled metal and As mobilisation, dispersal and storage in the Río Guadiamar, SW Spain and its implications for long-term contaminant fluxes to the Doñana wetlands.

Flood-related contaminant (As, Cd, Cu, Pb, Zn) remobilisation, dispersal and storage in the Río Guadiamar was investigated following the 1998 Aznalcóllar tailings dam failure, along with records of floodplain contaminant loading in the decades preceding the tailings release. A series of post-spill floods resulted in the transfer of vast quantities of sediment-borne heavy metals and As towards the lower reaches of the Guadiamar and the borders of the Doñana National Park, but over-bank flood deposits collected between May 1999 and March 2002 show a systematic fall in contaminant concentrations following successive flood events. Geochemical improvements can largely be attributed to sediment mixing of contaminated and 'clean' material derived from calcareous catchment soils. Longer-term contaminant patterns in floodplain sediment cores show higher heavy metal and As loading rates operating before the opening of the Aznalcóllar pit in 1979 and in some instances pre-dating 1954. The remobilization and dispersal of historically contaminated alluvium in the upper Guadiamar means that the post-clean-up contaminant signature in flood-transported sediments largely reflects chronic, long-term metal mining in the Guadiamar catchment, rather than the acute effects of the Aznalcóllar spill. Generally results present a cautiously optimistic prognosis for the sensitive wetlands of Doñana, but high dissolved (aqueous) heavy metal (especially Cu and Zn) concentrations in the upper Guadiamar emphasise the need for addressing contaminant 'hotspots' in the region and for maintaining flow requirements for aquatic ecosystems. This study illustrates the importance of establishing antecedent geomorphological-geochemical conditions in a spill-impacted river system, both for assessing the impacts of a single catastrophic pollution event and for developing appropriate strategies for remediation.

Modelling spatial and temporal variations of annual suspended sediment yields from small agricultural catchments.

Estimates of sediment yield are important for ecological and geomorphological assessment of fluvial systems and for assessment of soil erosion within a catchment. Many regulatory frameworks, such as the Convention for the Protection of the Marine Environment of the North-East Atlantic, derived from the Oslo and Paris Commissions (OSPAR) require reporting of annual sediment fluxes. While they may be measured in large rivers, sediment flux is rarely measured in smaller rivers. Measurements of sediment transport at a national scale can be also challenging and therefore, sediment yield models are often utilised by water resource managers for the predictions of sediment yields in the ungauged catchments. Regression based models, calibrated to field measurements, can offer an advantage over complex and computational models due to their simplicity, easy access to input data and due to the additional insights into factors controlling sediment export in the study sites. While traditionally calibrated to long-term average values of sediment yields such predictions cannot represent temporal variations. This study addresses this issue in a novel way by taking account of the variation from year to year in hydrological variables in the developed models (using annual mean runoff, annual mean flow, flows exceeded in five percentage of the time (Q5) and seasonal rainfall estimated separately for each year of observations). Other parameters included in the models represent spatial differences influenced by factors such as soil properties (% poorly drained soils and % peaty soils), land-use (% pasture or % arable lands), channel slope (S1085) and drainage network properties (drainage density). Catchment descriptors together with year-specific hydrological variables can explain both spatial differences and inter-annual variability of suspended sediment yields. The methodology is demonstrated by deriving equations from Irish data-sets (compiled in this study) with the best model efficiency of 0.84 and best model fit of adjusted R2 of 0.82. Presented approach shows the potential for regression based models to model contemporary suspended sediment yields in small river systems.

Effects of insertion conditions on tissue strain and vascular damage during neuroprosthetic device insertion.

Long-term integration of neuroprosthetic devices is challenged by reactive responses that compromise the brain-device interface. The contribution of physical insertion parameters to immediate damage is not well described. We have developed an ex vivo preparation to capture real-time images of tissue deformation during device insertion using thick tissue slices from rat brains prepared with fluorescently labeled vasculature. Qualitative and quantitative assessments of damage were made for insertions using devices with different tip shapes inserted at different speeds. Direct damage to the vasculature included severing, rupturing and dragging, and was often observed several hundred micrometers from the insertion site. Slower insertions generally resulted in more vascular damage. Cortical surface features greatly affected insertion success; insertions attempted through pial blood vessels resulted in severe tissue compression. Automated image analysis techniques were developed to quantify tissue deformation and calculate mean effective strain. Quantitative measures demonstrated that, within the range of experimental conditions studied, faster insertion of sharp devices resulted in lower mean effective strain. Variability within each insertion condition indicates that multiple biological factors may influence insertion success. Multiple biological factors may contribute to tissue distortion, thus a wide variability was observed among insertions made under the same conditions.

Selection of cancer chemotherapy for a patient by an in vitro assay versus a clinician.

One hundred thirty-three patients with advanced metastatic cancer were randomized to receive single-agent chemotherapy selected by either a medical oncologist or an in vitro capillary cloning system. Thirty-six of the 65 patients (55%) who were randomly assigned to selection of a drug by the clinician actually received a drug; these patients were able to be evaluated for clinical response. Of these 36 patients, one had a partial tumor response (3%). Only 19 of the 68 patients (28%) who were randomly assigned to selection of a drug by the capillary system actually received a drug; these patients were able to be evaluated for clinical response. Of these 19 patients, four (21%) had partial tumor responses. In the assessable patients (36 in the clinician's choice group, 19 in the capillary cloning group), the partial response rate was superior for drug selection by the capillary cloning system (P = .04). For all patients randomly assigned to a group (65 in the clinician's choice group, 68 in the capillary cloning group), the response rate was not significantly different (1.5% and 5.9%, respectively; P = .37). When overall survival rates for patients in the two groups were compared, there was no difference. We conclude that drug sensitivity testing in capillary tubes can improve the response rate for patients with advanced malignancies. This improved response rate, however, does not translate into improved survival times for these patients.

Phase I and clinical pharmacology trial of crisnatol (BWA770U mesylate) using a monthly single-dose schedule.

Crisnatol is a novel lipophilic arylmethylaminopropanediol with significant antineoplastic activity in a variety of murine and human tumor models which functions as a DNA intercalator. In this Phase I trial, a 6-h infusion of the drug was administered i.v. in 700 to 1500 ml of 5% dextrose in water every 28 days. Eighty-five courses at doses of 7.5 to 516 mg/m2 were administered to 43 patients with refractory solid tumors. Reversible neurological toxicity was dose limiting at 516 mg/m2 and was manifested as somnolence, dizziness, blurred vision, unsteady gait, and alpha-slowing on electroencephalogram at the end of infusion. All neurological signs and symptoms were reversible. No hematological toxicity was observed. Other toxicities included phlebitis, mild to moderate nausea and vomiting, reversible sinus node arrest in one patient, and hypertension. Crisnatol plasma concentrations were determined by high-pressure liquid chromatography. After infusion, plasma concentrations declined biexponentially with a terminal t1/2 of 2.9 h. Using a two-compartment model, the mean apparent volume of distribution at steady state and total-body clearance were 58.8 liters/m2 and 18.3 liters/h/m2, respectively, indicative of extensive tissue distribution and rapid hepatic clearance. Peak plasma levels occurred at the end of infusion and correlated with the onset of neurological toxicity. The recommended Phase II dose for this schedule is 388 mg/m2.

Phase I and clinical pharmacology trial of 502U83 using a monthly single dose schedule.

502U83 is an arylmethylaminopropanediol derivative exhibiting significant antineoplastic activity in a number of murine and human tumor models. In this Phase I trial, a 1-h or 4-h infusion of the agent was administered i.v. in 250 ml of 5% dextrose in water every 28 days. Fifty-three courses at doses of 25 to 2000 mg/m2 were administered to 36 patients with refractory solid tumors. Prolongation of the PR, QRS, and QT intervals on electrocardiograms was dose limiting at 2000 mg/m2. This prolongation appeared dose related and was reversible upon discontinuation of the infusion. No hematological toxicity was observed. Other toxicities included only sporadic and mild to moderate nausea and vomiting. No tumor responses were noted. 502U83 plasma concentrations were determined by high-pressure liquid chromatography. Complete pharmacokinetic profiles were obtained for 21 of the 36 patients. After infusion, plasma concentrations declined in a biexponential or in a triexponential manner with a harmonic mean terminal t 1/2 of 8.83 h. Using a three-compartment model, the mean apparent volume of distribution at steady state and total-body clearance were 195 liters/m2 and 42.5 liters/h/m2, respectively, indicative of extensive tissue distribution. No correlation could be found between the pharmacokinetic parameters and prolongation of the cardiac conduction intervals. Because of the cardiac effects with the drug, the schedule of administration of 502U83 used in this study cannot be recommended.

Evaluating the relationship between biotic and sediment metrics using mesocosms and field studies.

An ongoing research challenge is the detection of biological responses to elevated sediment and the identification of sediment-specific bioassessment metrics to evaluate these biological responses. Laboratory mesocosms and field observations in rivers in Ireland were used to evaluate the relationship between a range of biological and sediment metrics and to assess which biological metrics were best at discerning the effects of excess sediment on macroinvertebrates. Results from the mesocosm study indicated a marked decrease in the abundance of sensitive taxa with increasing sediment surface cover. % EPT (Ephemeroptera, Plecoptera, Trichoptera) and % E abundances exhibited the strongest negative correlation with sediment surface cover in the mesocosm study. The field study revealed that % EPT abundance was most closely correlated with % sediment surface cover, explaining 13% of the variance in the biological metric. Both studies revealed weaker relationships with a number of other taxonomy-based metrics including total taxon abundance, total taxon richness and moderate relationships with the Proportion of Sediment-sensitive Invertebrates metric (PSI). All trait-based metrics were poorly correlated with sediment surface cover in the field study. In terms of sediment metrics, % surface cover was more closely related to biological metrics than either re-suspendable sediment or turbidity. These results indicate that % sediment surface cover and % EPT abundance may be useful metrics for assessing the effect of excessive sediment on macroinvertebrates. However, EPT metrics may not be specific to sediment impact and therefore when applied to rivers with multiple pressures should be combined with observations on sediment cover.

The impact of cattle access on ecological water quality in streams: Examples from agricultural catchments within Ireland.

Unrestricted cattle access to rivers and streams represent a potentially significant localised pressure on freshwater systems. However there is no consensus in the literature on the occurrence and extent of impact and limited research has examined the effects on aquatic biota in the humid temperate environment examined in the present study. Furthermore, this is one of the first times that research consider the potential for cattle access impacts in streams of varying water quality in Northern Europe. We investigated the effects of cattle access on macroinvertebrate communities and deposited fine sediment levels, in four rivers of high/good and four rivers of moderate water quality status which drain, low gradient, calcareous grassland catchments in Ireland. We assessed the temporal variability in macroinvertebrates communities across two seasons, spring and autumn. Site specific impacts were evident which appeared to be influenced by water quality status and season. All four high/good water status rivers revealed significant downstream changes in community structure and at least two univariate metrics (total richness and EPT richness together with taxon, E and EPT abundance). Two of the four moderate water status rivers showed significant changes in community structure, abundance and richness metrics and functional feeding groups driven in the main by downstream increases in collectors/gatherers, shredders and burrowing taxa. These two moderate water status rivers had high or prolonged livestock activity. In view of these findings, the potential for some of these sites to achieve at least high/good water quality status, as set out in the EU Water Framework Directive, may be compromised. The results presented highlight the need for additional research to further define the site specific factors and livestock management practices, under different discharge conditions, that increase the risk of impact on aquatic ecology due to these cattle-river interactions.

Two new fluorescent dyes applicable for visualization of fungal cell walls.

Two fluorophores, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, not heretofore reported upon are described as useful dyes of fungal cell walls, septa and bud scars examined microscopically. The dyes, depending on the filter sets used, yield fluorescently stained material generally in the blue to green and yellow to red wavelengths for Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, respectively. They provide an excellent alternative to the more commonly used fluorophore, Calcofluor White M2R. The two fluorophores, in addition to being used at various spectral wavelengths from mercury arc sources, can be used with laser sources providing 488 nm and 543 nm line wavelengths, common to most scanning confocal microscopes. Unlike Calcofluor, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B do not fade quickly when exposed to selected light wavelengths; however, like Calcofluors they are compatible with living fungal cells.

Chemical and biological investigations of a transformer accident at Binghamton, NY.

A transformer fire occurred in a state office building in Binghamton, NY on February 5, 1981. Particulates from inside surfaces of ceiling panels on 16 of the 17 floors had concentrations of polychlorinated dibenzofurans (PCDFs) ranging from less than 1 part per million (ppm) to 1200 ppm while polychlorinated biphenyl (PCB) concentrations varied from 28 ppm to 23,000 ppm. In spite of the wide variations in contaminant concentrations, complete analytical data from 11 floors showed that there was a consistent PCDF/PCB ratio (0.067 +/- 0.026) and also consistent PCDF isomer group distributions (tetra-CDFs, 33 +/- 5%; penta-CDFs, 40 +/- 3%; hexa-CDFs, 18 +/- 7%; hepta-CDFs, 6 +/- 3%). It was found that the particulate samples could be successfully ranked in order of their degree of chemical contamination by an in vitro bioassay. The bioassay was based on induction of keratinization or changes in morphology in mouse epithelial cells. Animal toxicology experiments were carried out with a soot sample containing a PCDF concentration which approximated the mean value found on the ceiling particulates. The single dose oral LD values of the soot and its benzene extract equivalent, each administered to female guinea pigs in 0.75% methyl cellulose, were 410 and 327 mg/kg, respectively. These results demonstrated that the soot matrix had virtually no effect on the toxicity of the chemical contaminants in the soot. Morphological alterations in liver tissues from animals receiving the soot were found after examination by electron and light microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of astroglial cell function on micro-patterned surfaces with specific geometric parameters.

Microcontact printing techniques were used to modify silicon substrates with arrays of hexagonal features of N1[3-(trimethoxysilyl) propyl]diethylenetriamine (DETA) surrounded by octadecyltrichlorosilane (OTS), which are hydrophilic, cell-adhesive and hydrophobic, non-adhesive organosilanes, respectively. In the presence of serum proteins, LRM55 cell adhesion and morphology on these modified surfaces were best correlated to the width of the cell-adhesive features. On surfaces modified with small (5 microm in width) cell-adhesive features, LRM55 cells elaborated only thin processes. As feature width was increased, cells on these surfaces exhibited increased cell spreading and elaborated wide processes. On surfaces modified with large (>35 microm in width) features, single cells adhered to and spread upon individual DETA features. In a similar fashion, LRM55 cell adhesion density increased with increasing feature width; this correlation could be represented by a simple, second-order relation, and was independent of all other measures of pattern geometry. The results of this study provide evidence that micro-patterning may be effective in controlling astrocyte interaction with implant materials.

Axonal outgrowth of hippocampal neurons on micro-scale networks of polylysine-conjugated laminin.

Microcontact printing was used to define an interconnected lattice network of polylysine-conjugated laminin, a protein-polypeptide ligate that is an effective promoter of neuron outgrowth on material surfaces. In the presence of serum proteins, rat hippocampal neurons selectively adhered to features of polylysine-conjugated laminin as narrow as 2.6 microm in width. Adhering neurons extended long axonal processes, which precisely followed and did not deviate from the prescribed patterns, demonstrating that neurons respond to this protein with high selectivity and that these techniques effectively provide long-range guidance of axonal outgrowth. Further examination of neuron response under serum-free cell culture conditions demonstrated that the outgrowth-promoting activity of polylysine-conjugated laminin was attributed to biologically active laminin. Together, these results demonstrate that polylysine-conjugated laminin provides for high-precision guidance of neuron attachment and axon outgrowth on material surfaces in a serum-independent manner. This ability to guide hippocampal neuron response in low-density, serum-free culture with high precision is valuable for the development of advanced, neuron-based devices.

Selective adhesion of astrocytes to surfaces modified with immobilized peptides.

Under serum-free conditions, rat skin fibroblasts, but not cortical astrocytes, selectively adhered to glass surfaces modified with the integrin-ligand peptide RGDS. In contrast, astrocytes, but not fibroblasts, exhibited enhanced adhesion onto substrates modified with KHIFSDDSSE, a peptide that mimics a homophilic binding domain of neural cell adhesion molecule (NCAM). Astrocyte and fibroblast adhesion onto substrates modified with the integrin ligands IKVAV and YIGSR as well as the control peptides RDGS and SEDSDKFISH were similar to that observed on aminophase glass (reference substrate). This study is the first to demonstrate the use of immobilized KHIFSDDSSE in selectively modulating astrocyte and fibroblast adhesion on material surfaces, potentially leading to materials that promote specific functions of cells involved in the response(s) of central nervous system tissues to injury. This information could be incorporated into novel biomaterials designed to improve the long-term performance of the next generation of neural prostheses.

Phorbol ester-stimulated stellation in primary cultures of astrocytes from different brain regions.

Stellation is the process by which astrocytes change from epithelial-like to process-bearing cells. Stellation occurs following activation of either cyclic AMP-dependent protein kinase or protein kinase C. This process occurs through tubulin-dependent rearrangement of the cytoskeleton. We have evaluated the ability of phorbol, 12-myristate, 13-acetate (PMA) to induce astrocyte stellation. Astrocytes from five brain regions (cerebellum, cerebral cortex, hippocampus, diencephalon, and brain-stem) were examined to determine if all astrocytes would exhibit similar responses to this activator of protein kinase C. Stellation was evaluated following cell fixation by either phase optics using conventional light microscopy, or scanning laser confocal light microscopy of cultures prepared using immunocytochemistry for tubulin and glial fibrillary acidic protein. Both the number of cells responding to PMA and the sensitivity to PMA varied for astrocytes from each brain region. PMA-induced stellation was most robust in cerebellar and brainstem astrocytes, with greater than 70% responding. Less than 40% of hippocampal and diencephalic astrocytes responded to PMA at the maximum dose (10(-5) M). PMA also induced different numbers of processes or branching patterns of processes on astrocytes from different brain regions. The protein kinase C induced stellation response in astrocytes supports the hypothesis that astrocytes contribute to neural plasticity.

Localizing sites of intradendritic electrophysiological recordings by confocal light microscopy.

Studies were undertaken to develop microscopic methods and imaging procedures that would permit identification of sites of intradendritic microelectrode recordings from pyramidal cells in hippocampal slice preparations. Intradendritic recordings were obtained with sharp microelectrodes filled with the dye lucifer yellow. Following a recording session a neuron was iontophoretically injected with the dye and imaged by fluorescence videomicroscopy. Images were stored on videotape for later analysis. They provided a record of the location of the microelectrode recording site. After withdrawal of the microelectrode, slices were processed histologically and imaged a second time with a Bio-Rad 600 confocal attachment on an Olympus BH-2 microscope. Confocal images provided detailed anatomical information in three dimensions. In most instances, a clear identification of the recording site was achieved by comparing video images containing the recording electrode and confocal images. Neurophysiological recordings obtained from proximal and distal apical dendrites were markedly different. Proximal dendritic recordings were similar to those obtained from pyramidal cell soma. However, distal dendrites were not electroresponsive when depolarized by intracellular current injection. The techniques described here, or variations that employ patch electrodes, could provide valuable information that should further an understanding of the properties of dendrites in the central nervous system.

Developments in three-dimensional stereo brightfield microscopy.

We present recent developments of a widefield computer/microscope system and image reconstruction algorithm for producing three-dimensional (3D) increased depth of field images in the form of brightfield stereo pairs of thick specimens. The theoretical principle of this image reconstruction technique is based on Weiner-type inverse filtering. A number of extensions and refinements to our previous work have included further testing of the system with a broader class of specimens and the implementation of several pragmatic refinements important for future 3D microscopy systems. These refinements include histogram modification routines for improving visualization, a preprocessing routine to eliminate edge artifacts due to circular convolution and other effects, stereo viewing angle optimization, a rule of thumb estimate for the axial sampling rate, and incorporation of a variation of the Fast Fourier Transform and filtering operations that significantly reduce computational time. Images of spyrogyra, neonatal rat hippocampal neurons, and cervical/vaginal cell smears are presented to show the utility of these methods for 3D visualization. The primary advantages of these methods are that they operate with an ordinary transmitted light microscope and are inexpensively implemented on a personal computer with reasonable computation time.

Three-dimensional imaging and image analysis of hippocampal neurons: confocal and digitally enhanced wide field microscopy.

The microscopy of biological specimens has traditionally been a two-dimensional imaging method for analyzing what are in reality three-dimensional (3-D) objects. This has been a major limitation of the application of one of science's most widely used tools. Nowhere has this limitation been more acute than in neurobiology, which is dominated by the necessity of understanding both large- and small-scale 3-D anatomy. Fortunately, recent advances in optical instrumentation and computational methods have provided the means for retrieving the third dimension, making full 3-D microscopic imaging possible. Optical designs have concentrated on the confocal imaging mode while computational methods have made 3-D imaging possible with wide field microscopes using deconvolution methods. This work presents a brief review of these methods, especially as applied to neurobiology, and data using both approaches. Specimens several hundred micrometers thick can be sampled allowing essentially intact neurons to be imaged. These neurons or selected components can be contrasted with either fluorescent, absorption, or reflection stains. Image analysis in 3-D is as important as visualization in 3-D. Automated methods of cell counting and analysis by nuclear detection as well as tracing of individual neurons are presented.