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PubMed2005

Characterizing the three-dimensional organization of telomeres.

BACKGROUND: Quantitative analysis can be used in combination with fluorescence microscopy. Although the human eye is able to obtain good qualitative results, when analyzing the spatial organization of telomeres in interphase nuclei, there is a need for quantitative results based on image analysis. METHODS: We developed a tool for analyzing three-dimensional images of telomeres stained by fluorescence in situ hybridization in interphase nuclei with DNA counterstained with 4',6-diamidino-2-phenylindole. After deconvolution of the image, we segmented individual telomeres. From the location of the telomeres we derived a distribution parameter rhoT, which indicated whether the telomeres were in a disk (rhoT >> 1) or not (rhoT approximately 1). We sorted mouse lymphocyte nuclei and measured rhoT. We also performed a bromodeoxyuridine synchronous cell sorting experiment on live cells and measured rhoT at several instances. RESULTS: Measuring rhoT for nuclei in G0/G1, S, and G2 produced 1.4 +/- 0.1, 1.5 +/- 0.2, and 14 +/- 2, respectively, showing a significant difference between G2 and G0/G1 or S. For the bromodeoxyuridine synchronous cell sorting experiment, we found a cell cycle dependency of rhoT and a correlation between rhoT and an observer. CONCLUSIONS: In this study we present a quantitative method to characterize the organization of telomeres using three-dimensional imaging, image processing, and image analysis.

Algorithms↗

PubMed2005

Flow cytometric analysis of fluorescence in situ hybridization with dye dilution and DNA staining (flow-FISH-DDD) to determine telomere length dynamics in proliferating cells.

BACKGROUND: Telomeres shorten during DNA replication; extensive erosion of telomeres likely promotes replicative senescence and chromosomal instability. Telomere length in individual cells has been quantified by flow cytometric analysis of fluorescence in situ hybridization (flow-FISH). To determine the rate of telomere attrition (telomere erosion per cell division), we combined flow-FISH with dye dilution and DNA staining (flow-FISH-DDD) and measured telomere-specific fluorescence in proliferating cells identified by cell generation and cell cycle phase. METHODS: Peripheral blood mononuclear cells (PBMC) were stained with the cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with phytohemagglutinin (PHA), grown for 5-6 days, hybridized with a telomere sequence-specific peptide nucleic acid fluorescent probe (PNA-Cy5), counterstained with DAPI, and analyzed by flow cytometry. The cell cycle distribution and cell division generations were respectively identified by analysis of DAPI emission and deconvolution of CFSE emission, and Cy5 emission was used to determine telomere-specific fluorescence, an indicator of telomere length, in each cell. RESULTS: In stimulated PBMC, in each cell cycle phase, the telomere-specific fluorescence diminished with increasing cell generation. The rate of decline of the telomere-specific fluorescence per cell generation did not significantly differ between cell cycle phases. CONCLUSIONS: Application of flow-FISH-DDD to measure mean telomere length and the rate of telomere attrition in proliferating cells may find use in studies of ageing and disease, the effects of telomere-modifying agents, and variability between individuals.

Carbocyanines↗

PubMed1995

Uses of digital image analysis in electrophoresis.

The information in electrophoretic gels can be extracted visually, or by using specialised computer hardware and software which treat the gels as digital images. We discuss issues which arise in applying digital image analysis to electrophoresis, and comment on the range of computer packages available. We also illustrate how image analysis can make gel interpretation easier and more reliable. Methods covered include gel registration and warping, mathematical morphology and deconvolution to sharpen images.

Computers↗

PubMed2004

Capillary electrophoresis coupled to mass spectrometer for automated and robust polypeptide determination in body fluids for clinical use.

We describe the application of capillary electrophoresis (CE) coupled on-line to an electrospray ionization-time of flight-mass spectrometer (ESI-TOF-MS) to the analysis of human urine and serum for the identification of biomarkers for clinical diagnostics. CE-MS led to display > 1000 polypeptides present in complex biological samples within 45-60 min in a single analysis run. To extract the information of the CE-MS spectra in a timely fashion, a software was designed to automatically deconvolute and normalize the spectra. Both urine and serum contain several hundred polypeptides in samples from healthy individuals. Hence, it is possible to establish typical "normal urine" or "normal serum" polypeptide patterns. Samples from patients with different diseases display polypeptide patterns that differ significantly from those obtained from healthy individuals. Examining series of patients with the same disease allowed the establishment of polypeptide patterns typical for specific diseases. This permits the search for marker peptides specific for diseases. The data indicate that a single polypeptide present in all patients with the same disease, but absent in all healthy control individuals does not exist. The combination of several polypeptides found in either urine or serum or both are forming a specific pattern, which is indicative not only for the particular disease, but also for the stage of disease. CE-MS detects many polypeptides in single samples and the application of the software to the search of identical polypeptides excreted in urine allows the unbiased diagnosis based on a pattern and does not rely on single disease markers.

Automation↗

PubMed2005

Mass mapping of cancer cell lysates using two-dimensional liquid separations, electrospray-time of flight-mass spectrometry, and automated data processing.

Intact protein masses from immortal, nontransformed MCF10A, a human breast epithelial cell line, and its malignant derivative MCF10CA1a.cl1 have been mapped using a combination of all-liquid separations and automated data interpretation. Preparative liquid isoelectric focusing combined with nonporous silica reverse-phase high-performance liquid chromatography allows efficient separation of a large number of proteins in complex mixtures such as whole-cell lysates. Molecular weight determination of these proteins is achieved using electrospray-time of flight-mass spectrometry, however, manual data analysis for these separations is both complex and time-consuming. Protein mass mapping can be significantly enhanced by automating deconvolution functions typically performed manually, with resulting reductions in hands-on analysis time from 20-30 h per chromatogram to approximately 15 min. This reduction in analysis time allows for rapid screening of cancer cell lines for potential biomarkers over a wider pI range than would otherwise be possible.

Biomarkers, Tumor↗

PubMed2005

Protein profiling by capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry.

An automated system for intact protein analysis is described that combines capillary isoelectric focusing (CIEF), reversed-phase liquid chromatography (RPLC), and electrospray ionization-mass spectrometry (ESI-MS). Performance is demonstrated with a complex yeast enzyme concentrate. CIEF is performed with a microdialysis membrane-based cathodic cell that permits pI fractions to be sampled and stored for subsequent LC-MS analysis. A total of 50 microg protein is loaded onto the capillary. Ten fractions are stored which span the pI range 3-10. Each fraction is subsequently cleaned on a reversed-phase trap column and then characterized by LC-MS. MaxEnt1 is used to deconvolute the raw mass spectra to obtain the molecular weight (MW) of intact proteins/peptides in the sample. A two-dimensional display of pI vs. MW is illustrated for the 500 most prevalent species as identified by MaxEnt1.

Chromatography, Liquid↗

PubMed2005

Astrocytic exocytosis vesicles and glutamate: a high-resolution immunofluorescence study.

Physiological evidence has demonstrated that cultured astrocytes can release glutamate via Ca2+-dependent mechanisms. Also, glutamate released from astrocytes in the hippocampal slice interferes with synaptic neurotransmission. Since these observations suggest vesicular glutamate release from astrocytes, the presence of glutamate-containing exocytosis vesicles was investigated. We applied immunofluorescence techniques combined with high-performance deconvolution microscopy, which yields a resolution of <200 nm and permits evaluation of double labeling in individual vesicles. Using a well-characterized anti-glutamate antiserum and parameters minimizing fixative-induced autofluorescence, glutamate-immunoreactive (ir) puncta were found all over the astrocyte but were conspicuously dense at the cell boundary and in filopodia. Images were very similar with antibodies against vesicular glutamate transporters (vGluT1 and vGluT2). Labeling for the exocytosis markers rab3, synaptophysin, or synaptobrevin was also punctate, particularly dense at the cell boundary, but disappearing toward the perinuclear region. Sections of the cell boundary were delineated by rab3 immunoreactivity. In double-labeled cells, vesicular colocalization of glutamate and any of the exocytosis markers was frequent in filopodia and at the cell boundary. Within the cell, single-labeled glutamate-ir vesicles prevailed; double-labeled vesicles were infrequently present. By resolving single vesicles, in cultured astrocytes we visualize glutamate-containing vesicles, vesicles displaying vGluT1 or vGluT2, and exocytosis vesicles displaying glutamate-ir. This may provide the morphological correlate of Ca2+-dependent glutamate release from astrocytes, possibly occurring at defined sections of the cell membrane and at filopodia. However, since vGluTs and exocytosis markers are classically restricted to nerve terminals in the CNS, glutamate release from astrocytes in the CNS remains to be studied.

Animals↗

PubMed1993

Microtubule-dependent transport of bile salts through hepatocytes: cholic vs. taurocholic acid.

Studies with taurine-conjugated bile salts have demonstrated two pathways for hepatocellular delivery of bile salts to bile: a cytosolic, microtubule-independent pathway and a membrane-based, microtubule-dependent pathway. However, a significant portion of circulating bile salts may be unconjugated. To determine whether free bile salts utilize similar pathways, we examined the effect of colchicine on the biliary excretion of intravenously administered cholic acid and taurocholate in intact rats. Basal rats were pretreated with low-dose colchicine or its inactive isomer, lumicolchicine, 1 hr before placement of intravenous and biliary cannulas and 2.75 hr before intravenous injection of [14C]cholic acid and [3H]taurocholate. Superfused rats were prepared as above but with intravenous infusion of taurocholate at 200 nmol/min.100 gm beginning 0.75 hr before [14C]cholic acid/[3H]taurocholate injection. Depleted/reinfused rats were subjected to biliary diversion for 20 hr before colchicine or lumicolchicine pretreatment, infusion of taurocholate and [14C]cholic acid/[3H]taurocholate injection. In each group, biliary excretion of [14C]taurocholate and [3H]taurocholate was inhibited equally by colchicine; for peak excretion rates the respective inhibition values were 33% and 35% in basal rats, 63% and 65% in superfused rats, and 74% and 76% in depleted/reinfused rats. Biliary excretion of [14C]taurocholate occurred consistently later than excretion of [3H]taurocholate, and maximal rates of excretion were reduced. In contrast, plasma uptake rates of [14C]cholic acid and [3H]taurocholate were essentially the same in depleted/reinfused rats. Deconvolution analysis of [14C]taurocholate vs. [3H]taurocholate biliary excretion curves revealed no significant differences among experimental groups. We conclude that conversion of [14C]cholic acid to [14C]taurocholate slightly retards its biliary excretion and diminishes its peak excretion rate compared with exogenous [3H]taurocholate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Oral absorption characteristics and pharmacokinetics of colchicine in healthy volunteers after single and multiple doses.

Colchicine is an antimitotic drug used to treat gout and familial Mediterranean fever. Absolute bioavailability, pharmacokinetics, and absorption characteristics of colchicine after single 1.0-mg doses in oral solution or tablet form or 0.5-mg intravenous doses were compared in 6 subjects. This study was combined with 14 days of multiple-dose administration of 1.0-mg colchicine tablets in 6 subjects. Serial blood samples were collected for 48 hours after administration of single doses and for 120 hours after the last dose in the multiple-dose regimen. Plasma colchicine profiles as measured by radioimmunoassay were analyzed using deconvolution and compartmental methods. After intravenous bolus injection of colchicine, the area under the concentration-time curve (AUC) was 61.2 +/- 12.7 ng.hr/mL, steady-state volume of distribution (Vss) was 419 +/- 95 L, systemic clearance (Cl) was 8.5 +/- 1.8 L/hr, and the terminal half-life (t1/2) was 57.8 +/- 10.7 hours. After oral administration in solution form, peak plasma concentrations (Cmax) of 6.50 +/- 1.03 ng/mL were reached at time (tmax) 1.07 +/- 0.55 hours, with a rate of 0.109 +/- 0.024 hr-1 (Cmax/AUC); bioavailability was 47 +/- 14%. Oral tablets yielded similar Cmax, tmax, and Cmax/AUC values, but AUC was significantly lower. Most participants exhibited a secondary peak within 6 hours of administration, possibly in relation to a second absorption site or enterohepatic recirculation. This second absorption process was significantly longer than the first one, and accounted for a similar amount of colchicine absorbed. From the multiple-dose study, a model including an alteration of colchicine absorption due to possible drug-induced gastrointestinal modifications allowed better determination of steady-state plasma concentrations of colchicine.

Absorption↗

PubMed1997

Transdermal testosterone administration in hypogonadal men: comparison of pharmacokinetics at different sites of application and at the first and fifth days of application.

In this study of 13 hypogonadal men (25-69 years of age), three open-label, randomized treatments were administered to determine the pharmacokinetics of serum testosterone after application of an investigational testosterone transdermal system to the upper buttocks, upper arm, and back. Testosterone in vivo input kinetics profiles were estimated by DeMonS, a recently developed numerical deconvolution method for estimating drug absorption at different time intervals and/or drug disposition model parameters, and compared on the first and fifth days of system application. Area under the concentration-time curve from 0 to 27 hours (AUC0-27) values for testosterone after one-day applications to the upper buttocks, upper arm, and back were 9,560 ng.hr/dL, 8,651 ng.hr/dL, and 8,988 ng.hr/dL, respectively. Maximum observed concentration (Cmax) values were 482 ng/dL, 462 ng/dL, and 499 ng/dL, respectively. Serum testosterone concentrations were equivalent to each other, and Cmax values fell within the normal range. No drug accumulation was seen with repeated dosing over 5 days.

Administration, Cutaneous↗

PubMed1997

Testosterone pharmacokinetics after application of an investigational transdermal system in hypogonadal men.

This open-label, randomized, placebo lead-in, three-treatment crossover study in 19 hypogonadal men (27-82 years of age) evaluated dose proportionality of serum testosterone concentrations with application of one or two investigational transdermal testosterone systems for application to the arm or torso. Testosterone in vivo kinetics profiles were determined using DeMonS, a recently developed numerical deconvolution method that estimates drug absorption at different time intervals and/or drug disposition model parameters. After application of the investigational transdermal systems, the mean serum testosterone, dihydrotestosterone, estradiol, and free testosterone concentrations were elevated to normal levels. Treatment allowed approximation of the normal circadian pattern of endogenous testosterone secretion, and the increase in serum testosterone concentrations was proportional to the surface area of systems applied. The investigational transdermal system provided effective testosterone replacement therapy as judged by pharmacokinetic parameters.

Administration, Cutaneous↗

PubMedDate not supplied

Appraising the instantaneous secretory rates of luteinizing hormone and testosterone in response to selective mu opiate receptor blockade in late pubertal boys.

The pulsatile properties of gonadotropin and testosterone release were examined before and after chronic mu opiate receptor blockade with naltrexone, 50 mg every other day, in four normal boys in late puberty (ages 14 8/12 to 15 1/12 years). The nature of spontaneous secretory events was appraised for immunoactive LH and testosterone in blood withdrawn every 20 minutes for 24 hours, using a novel, discrete deconvolution algorithm to estimate apparent instantaneous secretory rates. The application of this methodology revealed that the frequency of discrete LH instantaneous secretory rates increased after mu opiate receptor blockade (P = 0.011). More strikingly, all parameters of testosterone secretory events responded significantly to mu opiate receptor blockade, including increases in mean estimated secretory rate (+47%, P = 0.02), testosterone pulse frequency (+ 64%, P less than 0.001) and amplitude (+ 20%, P = 0.027). Correspondingly, decreases in testosterone interpulse secretory intervals (-35%, P = 0.001), secretory pulse duration (-19%, P = 0.042) and interpulse valley duration (-35%, P = 0.006) also were noted. There was a prominent diurnal rhythm in testosterone secretion with maximal values in the morning and late evening, and marked reductions in the afternoon, sometimes to prepubertal levels. This variation in the testosterone secretory profile paralleled that of LH. In response to naltrexone, the FSH concentration series showed a significant increase in the mean FSH concentration (+ 18%) P = 0.003) and mean peak amplitude (+ 15%, P = 0.002). These data provide indirect evidence of functional coupling of the opiate system with the hypothalamic GnRH pulse generator.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMedDate not supplied

24-hour pulsatile and circadian patterns of cortisol secretion in alcoholic men.

Pulsatile and circadian patterns of cortisol secretion during acute (3 to 16 days) and chronic (29 to 39 days) abstinence were examined in alcoholic men with no clinical or laboratory evidence of hepatic dysfunction or nutritional deficiencies. Mean and integrated 24-hour serum concentrations of cortisol determined by sampling the blood every 20 minutes over a 24-hour period were increased in six out of 10 alcoholic subjects during acute abstinence when compared with normal controls. Sustained abstinence in seven subjects with follow-up studies caused significant decreases in the mean maximal cortisol peak amplitude (13 +/- 1.0 SEM acutely vs. 10.3 +/- 0.52 micrograms/dl follow-up; P = 0.01), mean 24-hour serum cortisol concentrations (10.9 micrograms/dl +/- 1.2 vs. 8.5 micrograms/dl +/- 0.26; P = 0.047), interpulse valley mean (9.3 micrograms/dl +/- 0.88 vs. 6.5 micrograms/dl +/- 0.34; P = 0.007), and valley nadir (7.9 micrograms/dl +/- 0.69 vs. 5.4 micrograms/dl +/- 0.30; P = 0.0036) concentrations. Cortisol pulse frequency was normal. Although circadian cortisol rhythmicity was maintained in alcoholics, the timing of the circadian acrophase was delayed significantly (P = 0.006) during acute abstinence (1022 [clocktime] +/- 34 min) as compared with normal controls (0743 [clocktime] +/- 34 min), and the amplitude of circadian cortisol rhythms exceeded normal in five of 10 alcoholics. Analysis of data in one alcoholic subject by a new multiparameter deconvolution method demonstrated increases in secretory burst amplitude (0.64 microgram/dl +/- 0.08 SD), mass of cortisol released per burst (9.8 micrograms/dl +/- 1.2 SD), and daily endogenous cortisol production rate (22 mg +/- 2.4 SD) during acute abstinence. These values were statistically different when compared with seven normal controls and the subjects' values during sustained abstinence (P less than 0.02). In conclusion, the results of the present study suggest increased daily production of cortisol as a possible mechanism underlying the elevated serum cortisol concentrations in chronic alcoholics during acute abstinence. This abnormality is shown to be reversible with sustained abstinence from alcohol.

Adult↗

PubMedDate not supplied

Discovery and design of novel inhibitors of botulinus neurotoxin A: targeted 'hinge' peptide libraries.

Intoxication by the zinc protease botulinus neurotoxin A (BoNT-A) results from cleavage of a single Q-R bond in the neuronal protein SNAP-25, which disables the docking mechanism required for neurotransmitter release. In the present study, potential inhibitors of BoNT-A were assessed from their effects on the BoNT-A cleavage of a synthetic 17-mer peptide (SNAP-25, residues 187-203) spanning the Q-R cleavage site. Compounds that inhibited BoNT-A included thiols (zinc chelators) such as dithiothreitol, dimercaptopropanesulfonic acid, mercaptosuccinic acid and captopril. In addition, compounds containing multiple acidic functions, such as the SNARE motif V2 (ELDDRADALQ), the tripeptide Glu-Glu-Glu and the steroid glycoside glycyrrhizic acid, were effective inhibitors. 'Hinge' peptide mini-libraries (PMLs) having the structure acetyl-X(1)-X(2)-linker-X(3)-X(4)-NH(2) or X(1)-X(2)-linker-X(3), where X(1)-X(4) were mixtures of selected amino acids and the flexible linker was 4-aminobutyric acid, also provided effective inhibition. Targeted PMLs containing the acidic amino acids Asp and Glu, the scissile-bond amino acids Gln and Arg and the zinc chelators His and Cys produced pronounced inhibition of BoNT-A. Deconvolution of these libraries will provide novel ligands with improved inhibitory potency as leads in the design of peptide mimetics to treat BoNT poisoning.

Antitoxins↗

PubMed1993

Osseous implant for studies of biomaterials using an in vivo electrochemical transducer.

The in vitro and in vivo electrochemical behavior of commercially pure titanium (cp Ti) was characterized using a specialized osseous implant in conjunction with electrochemical impedance spectroscopy (EIS) measurement techniques. Studies performed in vitro were used to verify the operation of the transducer and develop methods of deconvoluting EIS data. This method was subsequently used to describe an electrochemical equivalent circuit model of the surface oxide and electrical double-layer capacitance of cp Ti in the endogenous electrolyte found in the medullary compartment of a baboon tibia. Kinetic profiles of the double-layer capacitance and the polarization resistance were constructed from multiple in vitro and in vivo EIS measurements performed over 60 min at 0 V (reference Ag/AgCI) conditioning potential. The profiles demonstrated that the growth of surface oxides was biphasic, with rapid decrease in the double-layer capacitance occurring within 20 min and reaching steady-state conditions at approximately 40 min. These data suggested that a passive, stable biofilm formed on the cp Ti surface in vivo and in vitro.

Animals↗

PubMed2003

Lamellipodial motility in wounded endothelial cells exposed to physiologic flow is associated with different patterns of beta1-integrin and vinculin localization.

Integrins- and cytoskeletal-associated focal adhesion proteins may participate in the process of endothelial wound closure, but their relationship in these wounds and in the presence of shear forces has not been defined. The goal in this study was to test the hypotheses that (1) modulation of beta(1)-integrin in human coronary artery endothelial cells (HCAEC) would alter endothelial wound closure under shear stress, and (2) beta(1)-integrin association with vinculin would be necessary for mediating this closure. HCAEC monolayers were pre-conditioned to attain alignment by shearing at 12 dynes/cm(2) for 18 h in a parallel-plate flow chamber. Subsequently, they were divided into three groups: (a) control, (b) treated with anti-beta(1)-integrin adhesion blocking antibody, or (c) treated with anti-beta(1)-integrin adhesion promoting antibody. Next, the monolayers were wounded with a metal spatula, and re-sheared at 20 dynes/cm(2) or left static. Time-lapse imaging and deconvolution microscopy were then performed for 3 h. Immunocytochemistry for beta(1)-integrin expression and vinculin was performed on all wounded monolayers. Under shear stress, vinculin localized to the ends of stress fibers, while beta(1)-integrin took on an intracellular macroaggregate appearance. Treatment with anti-beta(1)-integrin adhesion blocking antibody enhanced wound closure, left the vinculin staining at the lamellipodial tips unchanged, but was associated with beta(1)-integrin staining at the lateral cell edges. Treatment with the anti-beta(1)-integrin adhesion promoting antibody retarded wound closure, increased vinculin staining at cell-cell junctions, and was associated with a fibrillar pattern of beta(1)-integrin staining. Modulation of beta(1)-integrin and changes in beta(1)-integrin and vinculin localization may further our understanding of laminar shear stress-induced endothelial repair in the coronary circulation.

Antibodies, Monoclonal↗

PubMed2006

Loss of mitochondrial membrane potential is associated with increase in mitochondrial volume: physiological role in neurones.

Mitochondrial volume homeostasis is a housekeeping cellular function, thought to help regulate oxidative capacity, apoptosis, and mechanical signaling. The volume is mainly regulated by potassium flux into and out of the matrix and controlled by the electrochemical potential. Mitochondrial depolarization will therefore affect this flux but studies showing how have not been consistent, and it is unclear what mitochondrial volume changes also occur. The aim of the present study was to investigate mitochondrial volume changes in permeabilized neurons under various bioenergetic conditions using deconvolution confocal microscopy. Under control conditions, mitochondria in situ appeared rod-shaped with mean length, surface area, and volume values of 2.29+/-0.10 microm, 1.41+/-0.10 microm2, and 0.062+/-0.006 microm3, respectively (n=42). Valinomycin, a K+-selective ionophore, increased mitochondrial volume by 63+/-22%, although surface area was almost unchanged because mitochondrial shape became more spherical. Pinacidil, an opener of mitochondrial ATP-dependent channels, produced similar effects, although some mitochondria were insensitive to its action. Mitochondrial depolarization with the protonophore FCCP, or with respiratory chain inhibitors antimycin and sodium azide was associated with a considerable increase in mitochondrial volume (by 75%-140%). Effects of mitochondrial modulators were also studied in intact neurones. Tracking of single mitochondria showed that during 65+/-2% of their time, mitochondria were motile with an average velocity of 0.19+/-0.01 microm/s. Antimycin, azide, and FCCP induced mitochondrial swelling and significantly decreased mitochondrial motility. In the presence of pinacidil, swollen mitochondria had reduced their motility, although mitochondria with normal volume stayed motile. These data show that mitochondrial depolarization was followed by significant swelling, which, in turn, impaired mitochondrial trafficking.

Animals↗

PubMed2003

Three-dimensional capillary geometry in gut tissue.

Accurate characterization of capillary geometry is of the utmost importance for physiological tissue studies such as oxygen transport. We show that 3D microscopy can be used to measure tissue capillary geometry both in normal and disease states. We imaged fluorescently labeled gut mucosa capillary beds of three control rats and three rats 4 hours after i.p. injection of 9 mg/kg endotoxin. We used serial optical sectioning microscopy coupled with deconvolution to reconstruct 3D capillary geometry. Theoretical point spread functions accounting for depth into the specimen resulted in better reconstructions than experimentally measured point spread functions. We next derived the distribution of the shortest distances to the nearest capillary from all extravascular tissue voxels. In normal rats the shortest-distance distributions were remarkably constant despite widely varying capillary geometry. Furthermore, the mean of the shortest-distance distributions increased significantly for endotoxemic rats (4.8+/-0.4 microm) compared to controls (4.3+/-0.3 microm, P<0.05). Hence, serial sectioning microscopy provides an accurate venue for measuring physiologically relevant 3D capillary structure.

Algorithms↗