Quantitative studies on ultrathin sections of bacteria infected with bacterio phage

A quantitative relationship has been established between the number of particles, for example bacteriophages, counted in ultrathin sections of bacteria and the total number present in the whole bacterial cells. The factor F relating particles counted per section with the total number of these particles per entire bacterium could be arrived at by two methods, which proved to give results in close agreement. The first involves knowledge of the average volume of a bacterial section in proportion to the average volume of a whole bacterium; if the mean number of appearances of the same particle on consecutive sections is also known, F may then be calculated. The thickness of sections and, therefore, their volume, as well as the average number of times a single particle is sectioned could be learned by examination of serial sections. By counting the relative number of T2 phage particles which had been intersected once or twice, and relating this proportion to the known phage dimensions, the thickness of the sections was determined to be about 400 A. The second measurement of F could be made in a particular case of late phage development where the number of particles per cell was countable or titratable directly in the bacterial lysate, this number being compared with the number seen in sections of the bacteria just before lysis. The different sources of errors are discussed. The statistical error is under 20 per cent, while the systematic errors are higher and cannot yet be indicated precisely. After a very cautious estimation of the upper limits, we can state, however, that the counts made with this method are certainly reliable to well within a factor of two.     

Considérations quantitatives sur des coupes ultraminces de bactéries infectées par un bactériophage

A quantitative relationship has been established between the number of particles, for example bacteriophages, counted in ultrathin sections of bacteria and the total number present in the whole bacterial cells. The factor F relating particles counted per section with the total number of these particles per entire bacterium could be arrived at by two methods, which proved to give results in close agreement. The first involves knowledge of the average volume of a bacterial section in proportion to the average volume of a whole bacterium; if the mean number of appearances of the same particle on consecutive sections is also known, F may then be calculated. The thickness of sections and, therefore, their volume, as well as the average number of times a single particle is sectioned could be learned by examination of serial sections. By counting the relative number of T2 phage particles which had been intersected once or twice, and relating this proportion to the known phage dimensions, the thickness of the sections was determined to be about 400 A. The second measurement of F could be made in a particular case of late phage development where the number of particles per cell was countable or titratable directly in the bacterial lysate, this number being compared with the number seen in sections of the bacteria just before lysis. The different sources of errors are discussed. The statistical error is under 20 per cent, while the systematic errors are higher and cannot yet be indicated precisely. After a very cautious estimation of the upper limits, we can state, however, that the counts made with this method are certainly reliable to well within a factor of two.     

Quantification of the human Sertoli cell population: its distribution, relation to germ cell numbers, and age-related decline

The human Sertoli cell population was characterized in 14 men by histometric analysis and by direct counts of nuclei in testicular homogenates. Testes obtained at autopsy were perfused with glutaraldehyde and embedded in Epon. Nucleolar and nuclear volumes were determined by the formula of a sphere given the diameter of the nucleoli or average diameter of nuclei measured at the height and width. Nuclear volume was also estimated by adding volumes of nuclear profiles in 0.5-micron serial sections. Sertoli cell number/g was calculated by the product of the percentage nucleoli or nuclei in the parenchyma, parenchymal volume, and histologic correction factor divided by the volume of a single nucleolus or nucleus. Also, Sertoli cell nuclei were counted directly in homogenates of fixed parenchyma. Number of Sertoli cells/g was similar (P greater than 0.05) whether determined by serial sections or in homogenates, but the estimate based on the nucleolar method was higher (P less than 0.01) and the nuclear measurement method was lower (P less than 0.01) than that for serial sections. A group of 37 men aged 20 to 48 yr had significantly (P less than 0.01) more Sertoli cells than did 34 men aged 50 to 85 yr. It is concluded that: 1) the homogenate method is valid for quantification of the Sertoli cell population, 2) Sertoli cells are evenly distributed in different regions of the testis, 3) the average human Sertoli cell supports relatively few germ cells, 4) the human Sertoli cell population declines with age, and 5) there is a significant relationship between sperm production rates and number of Sertoli cells.     

Stereologic estimation of mean nuclear volume in well-differentiated adenocarcinoma and carcinoma in situ of the gallbladder

OBJECTIVE: To investigate mean nuclear volume of cells in well-differentiated adenocarcinomas (20 cases) and carcinoma in situ (20 cases) of the gallbladder by the principle of estimation of the volume of particles with arbitrary shapes. STUDY DESIGN: Hematoxylin and eosin-stained, 4-micron-thick, vertical sections from formalin-fixed, paraffin-embedded tissue blocks were analyzed by using a projection microscope with a 100:1 oil immersion objective (NA 1.3); the final magnification was 2,500:1. The measurements were carried out in 10 microscopic fields for each slide. Mean nuclear volume was obtained by the stereologic method of point-sampled intercepts for vertical sections. RESULTS: Mean nuclear volume in well-differentiated adenocarcinomas (127.67 +/- 46.95 micron 3) was significantly larger than in carcinoma in situ (69.17 +/- 15.74 micron 3) (P < .000001). CONCLUSION: Stereologic estimation of mean nuclear volume may be helpful in the discrimination of malignant and borderline lesions of the gallbladder.     

Single-section counting error when distinguishing between primordial and early primary follicles in sections of rat ovary of different thickness

The number of primordial follicles within an ovary is frequently determined by counting 5, 7 or 10 microns thick sections and multiplying by the fraction of sections counted and a correction factor to adjust for duplicate counts. The objectives of the present study were: (i) to evaluate the accuracy of the correction factor developed by Abercrombie (1946); (ii) to evaluate the accuracy of the classification of primordial follicles from single tissue sections; and (iii) to determine the incorporation rate of 5-bromo-2-deoxyuridine into primordial follicles. In Expt 1, rat ovaries were sectioned at a thickness of 5, 7 or 10 microns. Primordial follicles were counted and classified across ten adjacent ovarian sections. The percentage of primordial follicles from single sections that were counted twice was 10, 9 and 2% in 5, 7 and 10 microns sections, respectively. This was lower than predicted by Abercrombie's method. The major error in counting from single sections was classification of early primary follicles as primordial follicles (55, 33 and 3% in 5, 7 and 10 microns sections, respectively). In Expt 2, a mean of 12 +/- 7% of primordial follicles incorporated 5-bromo-2-deoxyuridine after infusion for 7 days (four of seven rats had no labelled primordial follicles). In conclusion: (i) Abercrombie's correction factor should not be used for adjusting counts of follicles; (ii) evaluation of primordial follicles from single sections gives inaccurate counts and incorrect classification is of greater importance than duplicate counting, particularly in thinner sections; (iii) for evaluation of the number of follicles, 10 microns is the optimal thickness; and (iv) primordial follicles incorporated 5-bromo-2-deoxyuridine infrequently.     

Blockade of sphingosine-1-phosphate reduces macrophage influx and retinal and choroidal neovascularization

Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule that stimulates endothelial cell migration, proliferation, and survival in vitro, and tumor angiogenesis in vivo. In this study, we used a humanized monoclonal antibody (sonepcizumab) that selectively binds S1P to investigate its role in retinal and choroidal neovascularization (NV). Intraocular injection of sonepcizumab significantly reduced macrophage influx into ischemic retina and strongly suppressed retinal NV in mice with oxygen-induced ischemic retinopathy. In mice with laser-induced rupture sites in Bruch's membrane, intraocular injection of sonepcizumab significantly reduced the area of choroidal NV and concomitantly reduced fluorescein leakage from the remaining choroidal NV. Four weeks after intraocular injection of up to 1.8 mg of the sonepcizumab in non-human primates, electroretinograms and fluorescein angiograms were normal, and light microscopy of ocular sections showed no evidence of structural damage. These data show for the first time that S1P stimulates both choroidal and retinal NV and suggest that sonepcizumab could be considered for evaluation in patients with choroidal or retinal NV.     

An Ocular Grid-Planimeter Method for Enumerating Nerve Fibers

The estimation of numbers of nerve fibers in cross sections of peripheral nerves containing both fine and large fiber components can be accomplished by using an ocular grid and selectively counting a known area. With the use of a projecting apparatus and planimeter, the total cross section area is determined.

The following proportion expresses the principle involved:

total number of fibers in the nerve area of ass section of entire nerve number counted in the sample area area of sample

I f the planimeter calibration and the magnification of the tracing remain the same, a constant factor may be used for successive estimates. This factor is equal to the value of the planimeter reading of 1.000 divided by the area of the grid times the magnification squared. The final calculations are made by multiplying the number of fibers counted times the planimeter reading times the constant and dividing by the number of grid squares counted.

Counts of some nerves, using high magnification in enumerating the sample areas, can be finished in less than an hour after the preparation of slides. In comparing numbers obtained by complete counts with estimated numbers, the error was determined to be approximately ± 5%     

Quantitation of gold labeling and estimation of labeling efficiency with a stereological counting method.

The amount of immunolabeling over a cell compartment of an average cell was estimated by use of an adaptation of the double disector method introduced by Gundersen. The first and last sections of a stack of ultra-thin sections formed a disector in which cell number could be estimated and related to a defined reference volume to give the cell density. Another stack section, selected at random, was immunolabeled and the number of gold particles associated with unit volume reference space (gold "density") estimated in quadrats placed systematically across the section. The ratio of gold density to cell density was used to estimate the number of gold particles lying over a chosen compartment of an average cell, N(gold)/N(cell). Such estimates required neither cell volume nor section thickness measurement and were reproducible. By combining the number of gold particles per cell with estimates of the number of protein antigens per cell, the number of gold particles associated with each antigen could be found (labeling efficiency).     

Dermatan sulfates of normal and scarred fascia

We evaluated the composition of dermatan sulfates (DS) derived from 23 samples of normal and 23 samples of scarred fascia lata. We analyzed the molecular weight of intact DS chains and the length of chain regions comprising: (1) clusters of L-iduronate-containing disaccharides ("iduronic sections"); (2) clusters of D-glucuronate-containing disaccharides ("glucuronic sections"); and (3) copolymeric sections with both types of disaccharides. A portion of scarred fascia DS chains demonstrated higher molecular weight compared with those from normal tissue. Most disaccharides of DS chains derived from both fascia types form copolymeric segments - heterogeneous in size - with alternatively distributed single disaccharides with glucuronic residues and mainly single ones with iduronate. Only a small number of disaccharides form "glucuronic sections" of heterogeneous size or short "iduronic sections". However, the scarred fascia DS chains demonstrate an increased content of shorter "glucuronic sections" and shorter, often oversulfated, copolymeric segments. It seems that in normal fascia, the DS chain type with a single, long copolymeric region and a single, shorter "glucuronic section" is predominant, while in scarred tissue an increase in multidomain DS chain content may occur.     

Extended-gate FET-based enzyme sensor with ferrocenyl-alkanethiol modified gold sensing electrode

We developed a field-effect transistor (FET)-based enzyme sensor that detects an enzyme-catalyzed redox-reaction event as an interfacial potential change on an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. While the sensitivity of ion-sensitive FET (ISFET)-based enzyme sensors that detect an enzyme-catalyzed reaction as a local pH change are strongly affected by the buffer conditions such as pH and buffer capacity, the sensitivity of the proposed FET-based enzyme sensor is not affected by them in principle. The FET-based enzyme sensor consists of a detection part, which is an extended-gate FET sensor with an 11-FUT immobilized gold electrode, and an enzyme reaction part. The FET sensor detected the redox reaction of hexacyanoferrate ions, which are standard redox reagents of an enzymatic assay in blood tests, as a change in the interfacial potential of the 11-FUT modified gold electrode in accordance with the Nernstian response at a slope of 59 mV/decade at 25 degrees C. Also, the FET sensor had a dynamic range of more than five orders and showed no sensitivity to pH. A FET-based enzyme sensor for measuring cholesterol level was constructed by adding an enzyme reaction part, which contained cholesterol dehydrogenase and hexacyanoferrate (II)/(III) ions, on the 11-FUT modified gold electrode. Since the sensitivity of the FET sensor based on potentiometric detection was independent of the sample volume, the sample volume was easily reduced to 2.5 microL while maintaining the sensitivity. The FET-based enzyme sensor successfully detected a serum cholesterol level from 33 to 233 mg/dL at the Nernstian slope of 57 mV/decade.     

小鼠海马神经元冰冻切片和脑片培养方法的比较

目的:通过冰冻切片和脑片培养方式比较获得更适合脑片实验研究的方法。方法:分别运用急性切片和脑片培养方法,结合全细胞膜片钳技术比较两种脑片处理方法对小鼠海马神经元细胞形态、细胞膜封接难易程度、细胞电生理特性等的差异,获得更适合细胞研究的脑片获取方法。结果:冰冻切片方法切断部分神经纤维,脑片表层出现肿胀或坏死细胞,2-3层细胞可用于膜片钳记录,但不易封接破膜。脑片培养后可使纤维再生,整个脑片细胞形态清晰可见,容易封接破膜,电生理记录波形及基本特性与冰冻切片一致,但脑片培养方法的细胞突触后电流幅度更大、频率更高。结论:脑片培养可修复受损纤维和细胞膜柔韧性,且不改变膜特性,但脑片培养重建了一定数量的细胞间信号连接,使细胞反应性增强,脑片培养方法更适合脑片神经元研究。     

A Monte Carlo simulation of the determination of mean particle volume using the Cavalieri estimator

BACKGROUND: A common morphometric problem is the determination of an estimate of the size of biological particles obtained from measurements made on a sample of profiles observed in sections. Results are reported typically in terms of mean caliper diameter or mean volume of the particle. METHODS: We have investigated the use of the Cavalieri estimator for obtaining estimates of mean particle volume using a Monte Carlo simulation. Samples of spherical and ellipsoidal particles were generated by computer and serially sectioned at a fixed mean thickness with a small, imposed random variation. The area of each profile was determined and the volume of the particle was calculated according to the Cavalieri estimator. The influence on the estimate of the mean particle volume and its 95% confidence interval was evaluated for several variables: the shape of the particles, the standard deviation of the particle volume in the population, the section thickness, and the standard deviation of the section thickness. RESULTS: The results obtained with the Cavalieri estimator correspond favorably with those obtained with previously reported alternative methods. This leads to a recommendation for the strong consideration for the use of the Cavalieri estimator in cases in which it is technically feasible to obtain at least three sections through the individual particles. Graphs are provided, which relate the confidence interval for the mean volume to the number of particles measured.     

Fraction of neoplastic cells in breast cancer samples. Correction for the oncogene amplification test

OBJECTIVE: To develop the necessary correction for the polymerase chain reaction-based oncogene amplification test applied to paraffin-embedded tissue sections. STUDY DESIGN: The theoretical basis for making the correction in test results and for determining the neoplastic cell fraction was clarified, and a method for microscopic sampling was developed. The cell count bias was corrected by applying an equation by Ebbeson and Tang and using two adjacent tissue sections of different thickness, cut from the block from which DNA was isolated. RESULTS: There was no need to determine the absolute thicknesses of the sections. Section thickness, as a relevant feature, was cancelled out in developing the formula for estimating the cell fraction. The correction was applied in practice, and when tested on 10 breast cancer samples, the correlation between amplification estimates from two observers was good (r = .96). CONCLUSION: This method is valuable for interpreting the test results based on paraffin sections and for developing simpler quantitating methods for special test applications. The study also showed that amplifications may be more common than expected on the basis of the crude results of this type of test.     

Distinct parameters are involved in controlling the number of rounds of cell division in each tissue during ascidian embryogenesis.

We counted cell numbers during embryogenesis of the ascidian, Halocynthia roretzi, every hour. Cell numbers were determined by counting the numbers of nuclei in squashed embryos. The cell number of a larva just after hatching was approximately 3000. Our study addresses the question of what factors control the number of rounds of cell division during development. Three kinds of egg fragments were prepared by cutting unfertilized eggs to alter the volume of cytoplasm and the amount of DNA. After the egg fragments were fertilized, the cell numbers were estimated at the hatching stage. The cell numbers of the resulting larvae differed from those of normal larvae. Precursor blastomeres of various tissues were then isolated from normal and manipulated embryos, and cultured as partial embryos. The cell numbers of the resulting partial embryos were counted to estimate the number of cell divisions in each larval tissue. The results suggested that the number of cell divisions is controlled by a distinct mechanism in each tissue. We propose that the number of rounds of cell division during ascidian embryogenesis is controlled by three mechanisms: the first depending on the volume of cytoplasm; the second on the nucleo-cytoplasmic ratio; and the third depending on neither of these parameters. J. Exp. Zool. 284:379-391, 1999.     

Adjusting interphase FISH results in epithelial tissue sections to whole cell complement

OBJECTIVE: To derive an equation to compensate for the discrepancies between whole cell preparations and tissue sections for more accurate enumeration of fluorescence in situ hybridization (FISH) signals per cell. STUDY DESIGN: Mean centromere signal counts in touch preparations and corresponding 4-6-micron sections of paraffin-embedded tissue were calculated. Mean widest nuclear diameters were also determined from the tissue sections. The observed data were analyzed to define the volumetric relationships between tissue sections and whole cell preparations. RESULTS: Analysis of results from six lung specimens yielded an equation that approximates whole versus sectioned nuclear volume and permits accurate quantification of mean FISH signal count in histologic sections, as follows: [formula: see text].     

Two independent mechanisms for escaping epidermal growth factor- mediated growth inhibition in epidermal growth factor receptor- hyperproducing human tumor cells

Human squamous cell carcinoma cell lines often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGF. To investigate the mechanism of EGF-mediated inhibition of cell growth, variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGF receptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant isolated from Ca9-22 cells, CER-1, grew without being affected by EGF. CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicated an increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independent mechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.     

Split Nucleoli as A Source of Error in Nerve Cell Counts

The number of nerve cells in a given ganglion or nucleus is usually determined by counting the nucleoli in serial sections. The possibility that nucleoli may split and appear in more than one section is recognized as a source of error. A determination of the value of this error was made as follows; from nodose ganglia of four cats were cut serial transverse sections in which the sections varied in thickness. Thus from one ganglion, four sections were cut at 12 μ, then six at 9μ, and eight at 6μ. The process was repeated over and over until the entire ganglion was sectioned. The other ganglia were sectioned similarly. After mounting and staining, separate counts were made of the nucleoli of each given ganglion from the sections of different thicknesses. If nucleoli split according to theoretical expectations, the percentage of nucleoli split in thick sections should be less than the percentage split in thinner sections and the counts based on the sections of different thicknesses should vary accordingly. The results obtained indicate that the counts from thin sections do not differ appreciably from counts from much thicker sections, i. e., the thickness of the sections does not affect the count. It is, therefore, concluded that no correction should be made for split nucleoli if the sections are around 10 μ in thickness and none but distinct and definite nucleoli are counted.     

Novel efficient methods for measuring mesophyll anatomical characteristics from fresh thick sections using stereology and confocal microscopy: application on acid rain-treated Norway spruce needles

Recent design-based stereological methods that can be applied to thick sections cut in an arbitrary direction are presented and their implementation for measuring mesophyll anatomical characteristics is introduced. These methods use software-randomized virtual 3D probes, such as disector and fakir test probes, in stacks of optical sections acquired using confocal microscopy. They enable unbiased estimations of the mean mesophyll cell volume, mesophyll cell number in a needle, and for the first time an internal surface area of needles or other narrow leaves directly from the fresh tissue cross-sections cut using a hand microtome. Therefore, reliable results can be obtained much faster than when using a standard microtechnical preparation. The proposed methods were tested on Norway spruce needles affected for 1 year by acid rain treatment. The effect of acid rain resulted in changes of mesophyll parameters: the ratio of intercellular spaces per mesophyll cell volume increased, while needle internal surface area, total number of mesophyll cells, and number of mesophyll cells per unit volume of a needle decreased in the treated needles.     

Morphology and histochemistry of the adrenal medulla. I. Various types of primary catecholamine-storing cells in the mouse adrenal medulla.

Semithin sections (Araldite) of mouse adreno-medullary tissue were examined in the light microscope after perfusion fixation with glutaraldehyde, glutaraldehyde/formaldehyde or after freeze-drying followed by a treatment with hot formaldehyde gas. The following methods were employed: (i) aldehyde-induced fluorescence of catecholamines, (ii) Schmorl's ferric ferricyanide reaction, (iii) argentaffin reaction, and (iiii) staining with alkaline lead citrate followed by Timm's silver sulphide reaction. The correspondence of results obtained by the various methods was proven in consecutive sections or by successively applying different methods to identical sections. Four types of primary catecholamine-storing cells were identified. NA1 cells contain cytoplasmic granules up to 0.3 mum in diameter which stain black with ammoniacal silver and display a bright white to yellow fluorescence. NA2 cells show smaller cytoplasmic granules which stain brown with the argentaffin method and give white catecholamine fluorescence. NA3 cells appear yellow-earth after applying the argentaffin reaction and show greenish fluorescence. NA4 cells are hardly identified in the light microscope. These cells are significantly smaller than the above mentioned cells and characterized by a high nucleo-cytoplasmic ratio. They become straw coloured with ammoniacal silver and show greenish fluorescence. The argentaffin reaction was also used to identify these cells in semithin sections of glutaraldehyde/osmium tetroxide fixed material. The fine structure of the various noradrenalin-storing cells was studied in consecutive thin sections. NA1 cells were found to contain two populations of granules, the larger ones measuring between 300 and 350 nm, the smaller ones about 175 nm. The granules in NA2 cells correspond to this latter population (175 nm). NA3 cells contain an uniform granule population with a main diameter of 120 nm. The smallest granules are seen in NA4 cells being in the dimension of 80 nm. Granules in NA1 and NA2 cells show uniformly high density, whereas those in NA3 and NA4 cells display cores of varying density. Granules with moderately dense cores in NA3 and NA4 cells may represent partially emptied sites of noradrenalin storage or dopamin containing particles.     

Toxin detection by Si photosensitive biosensors with a new measurement scheme

We propose a new type of photosensitive biosensor with a CMOS compatible Si photodiode integrated circuit, for the high-sensitive detection of small mycotoxin molecules requiring competitive assay approach. In this work, a photodiode is connected to the gate of a field effect transistor (FET) so that the open circuit voltage (V(OC)) of the illuminated photodiode is transferred into the drain/source current (I(DS)) of the FET. The sensing scheme employs competitive binding of toxin molecules (within the sample solution) and toxin-BSA conjugates (immobilized on the photodiode surface) with Au-nanoparticle-labeled antibodies, followed by silver enhancement to generate opaque structures on the photodiode surface. By utilizing the non-linear dependence of the V(OC) on the light intensity, we can maintain a sufficiently high signal resolution at low toxin concentrations (with most of the incident light blocked) for the competitive assay. By monitoring the I(DS) of the FET whose gate is driven by the V(OC), quantitative detection of Aflatoxin B1 has been achieved in the range of 0-15ppb.