3D representation and analysis of enthesis morphology

This comparison of methods for assessing the development of muscle insertion sites, or entheses, suggests that three‐dimensional (3D) quantification of enthesis morphology can produce a picture of habitual muscle use patterns in a past population that is similar to one produced by ordinal scores for describing enthesis morphology. Upper limb skeletal elements (humeri, radii, and ulnae) from a sample of 24 middle‐aged adult males from the Pottery Mound site in New Mexico were analyzed for both fibrous and fibrocartilaginous enthesis development with three different methods: ordinal scores, two‐dimensional (2D) area measurements, and 3D surface areas. The methods were compared using tests for asymmetry and correlations among variables in each quantitative data set. 2D representations of enthesis area did not agree as closely as ordinal scores and 3D surface areas did regarding which entheses were significantly asymmetrical. There was significant correlation between 3D and 2D data, but correlation coefficients were not consistently high. Intraobserver error was also assessed for the 3D method. Cronbach's alpha values fell between 0.68 and 0.73, and error rates for all entheses fell between 10% and 15%. Marginally acceptable intraobserver error and the analytic versatility of 3D images encourage further investigation of using 3D scanning technology for quantifying enthesis development. Am J Phys Anthropol 152:417–424, 2013. © 2013 Wiley Periodicals, Inc.     

3-D reconstruction of biological objects using underwater video technique and image processing

This paper describes a 3-D reconstruction method which allows accurate measurements of volume, surface area and other morphometric measurements of three-dimensional biological objects, without removing them from the sea. It represents a novel approach based on multiple views (eight resulted to be sufficient) from underwater video images and a new image processing procedure (MOD3D), whose application has met the basic requirements (i.e. to work on images recorded in turbid waters, with nonuniform lighting, to investigate large areas and in reasonable time, etc.) imposed when operating in the marine environment with simple, easy-to-use and nonprofessional equipment. It is a noninvasive, nondestructive and in the field fast method, thus suitable for sampling also at relevant depth, whose applicability has specifically been set up for a range of growth forms from massive to submassive and irregularly shaped. The accuracy of the method was assessed using models with three levels of 3-D complexity: simple, moderate and complex morphology. A high accuracy of volume measurements made through MOD3D image analysis software was achieved when compared with the laboratory water displacement method, which represents the most accurate method for volume measurement, with an overall mean percent error of about 1.7% (S.D. 2.2%). For all three levels of morphologic complexity, no significant differences (p>0.05) were found. Volume measurements obtained in field based on geometric approximation resulted rough, with significant differences from the MOD3D values (p<0.05). The geometric approximation was lower than MOD3D for simple and moderate morphology, and variable for complex morphology. For all three models, MOD3D values for surface area computation were consistently lower (mean error 13%) than the foil-wrapping values (p<0.05), due to overlap error when foil wrapping. Two applications were made with the bryozoan Pentapora fascialis and the coral Cladocora caespitosa to quantify carbonate standing stock and biomass of these two carbonate framework builders, whose importance has been recently recognised among the temperate sublittoral benthic species. Time required for the 3-D reconstruction method (about 3 h) makes it suitable for routine application particularly for relatively large area investigations, with irregularly shaped objects on rough substrate and several biological objects within the area.     

Combined physical and chemical immobilization of glucose oxidase in alginate microspheres improves stability of encapsulation and activity

Chemical sensors utilizing immobilized enzymes and proteins are important for monitoring chemical processes and biological systems. In this study, calcium-cross-linked alginate hydrogel microspheres were fabricated as enzyme carriers by an emulsification technique. Glucose oxidase (GOx) was encapsulated in alginate microspheres using three different methods: physical entrapment (emulsion), chemical conjugation (conjugation), and a combination of physical entrapment and chemical conjugation (emulsion-conjugation). Nano-organized coatings were applied on alginate/GOx microspheres using the layer-by-layer self-assembly technique in order to stabilize the hydrogel/enzyme system under biological environment. The encapsulation of GOx and formation of nanofilm coating on alginate microspheres were verified with FTIR spectral analysis, zeta-potential analysis, and confocal laser scanning microscopy. To compare both the immobilization properties of enzyme encapsulation techniques and the influence of nanofilms with uncoated microspheres, the relationship between enzyme loading, release, and effective GOx activity (enzyme activity per unit protein loading) were studied over a period of four weeks. The results produced four key findings: (1) the emulsion-conjugation technique improved the stability of GOx in alginate microspheres compared to the emulsion technique, reducing the GOx leaching from microsphere from 50% to 17%; (2) the polyelectrolyte nanofilm coatings increased the GOx stability over time, but also reduced the effective GOx activity; (3) the effective GOx activity for the emulsion-conjugation technique (about 3.5 x 10(-)(5) AU microg(-)(1) s(-)(1)) was higher than that for other methods, and did not change significantly over four weeks; and (4) the GOx concentration, when compared after one week for microspheres with three bilayers of poly(allylamine hydrochloride)/sodium poly(styrene sulfonate) ({PAH/PSS}) coating, was highest for the emulsion-conjugation technique. As a result, the comparison of these three techniques showed the emulsion-conjugation technique to be a potentially effective and practical way to fabricate alginate/GOx microspheres for implantable glucose biosensor application.     

An alternative method of anthropometry of anterior cruciate ligament through 3-D digital image reconstruction

Accurate and flexible measurements of length, area, and volume are important in evaluation of the mechanical properties of soft tissue. Although a number of contact-based and non-contact techniques have been reported in the literature, due to a variety of reasons such as cost, complexity, and low accuracy, the research community has not adopted a standardized technique. In this paper, an alternative method of measuring the geometric parameters of cadaver anterior cruciate ligament (ACL) is presented. In this method, a 3-D scan of the ACL is constructed using a simple, commercially available, scanning system. The 3-D scan is then analyzed using the 3-D Doctor Software to extract important information regarding the length, cross-sectional area, and volume of the ACL. The accuracy and repeatability of measurements obtained by this method are acceptable and comparable to existing non-contact methods. The limitation of the method is that surface concavities cannot be detected. However, the non-contact optical method, described here, has inherent advantages over the existing methods: (1) it is inexpensive; (2) it allows the determination of area at any distance along the length of the tissue of interest; (3) all relevant information including minimum area is extracted from one single application of the method; (4) the volume can be calculated with a simple additional step of length measurement although, for accurate results, condylar blockage must be minimized by coring the ACL out. The entire process of scanning takes less than 30 min. This technique has the potential to become a standard method in anthropometry of soft tissue.     

Assessing the Geometric Structure of a White Clover (Trifolium repens L.) Canopy using3-D Digitising

A method was developed for assessing the three dimensional (3-D)geometric structure of white clover canopies. 3-D co-ordinatesof pre-defined points on leaves, petioles and stolons were measuredusing a Polhemus Fastrak electromagnetic 3-D digitiser. Digitisingprogressed downwards from the top of the canopy and plant partswere removed after they have been digitised. Leaflets were treatedas four quarter-ellipses, and petiole and stolons were treatedas cylinders. Leaf dimensions and areas calculated from 3-Dco-ordinates were within about 5% and 20% of direct measurementsmade with a ruler and a planimeter, respectively. Special softwareand freeware POV-Ray were used to reconstruct a virtual canopyfrom digitiser records and to calculate canopy characteristicssuch as leaf area index (LAI), petiole intersection area, andprofiles of leaflet areas and inclinations with height. It tookbetween 3 and 7 h to digitise 10 x 10 cm stands of clover andthe resulting information was considerably more comprehensiveand accurate than could have been obtained by the alternative‘point quadrat’ or ‘stratified clipping’methods.Copyright 2000 Annals of Botany Company White clover, Trifolium repens, geometric structure, leaf area, leaf angle, 3-D digitising     

Technical Note: 3D From Standard Digital Photography of Human Crania—A Preliminary Assessment

This study assessed three‐dimensional (3D) photogrammetry as a tool for capturing and quantifying human skull morphology. While virtual reconstruction with 3D surface scanning technology has become an accepted part of the paleoanthropologist's tool kit, recent advances in 3D photogrammetry make it a potential alternative to dedicated surface scanners. The principal advantages of photogrammetry are more rapid raw data collection, simplicity and portability of setup, and reduced equipment costs. We tested the precision and repeatability of 3D photogrammetry by comparing digital models of human crania reconstructed from conventional, 2D digital photographs to those generated using a 3D surface scanner. Overall, the photogrammetry and scanner meshes showed low degrees of deviation from one another. Surface area estimates derived from photogrammetry models tended to be slightly larger. Landmark configurations generally did not cluster together based upon whether the reconstruction was created with photogrammetry or surface scanning technology. Average deviations of landmark coordinates recorded on photogrammetry models were within the generally allowable range of error in osteometry. Thus, while dependent upon the needs of the particular research project, 3D photogrammetry appears to be a suitable, lower‐cost alternative to 3D imaging and scanning options. Am J Phys Anthropol 154:152–158, 2014. © 2014 Wiley Periodicals, Inc.     

A precise and non-destructive method to calculate the surface area in living scleractinian corals using X-ray computed tomography and 3D modeling

The surface area of corals represents a major reference parameter for the standardization of flux rates, for coral growth investigations, and for investigations of coral metabolism. The methods currently used to determine the surface area of corals are rather approximate approaches lacking accuracy, or are invasive and often destructive methods that are inapplicable for experiments involving living corals. This study introduces a novel precise and non-destructive technique to quantify surface area in living coral colonies by applying computed tomography (CT) and subsequent 3D reconstruction. Living coral colonies of different taxa were scanned by conventional medical CT either in air or in sea water. Resulting data volumes were processed by 3D modeling software providing realistic 3D coral skeleton surface reconstructions, thus enabling surface area measurements. Comparisons of CT datasets obtained from calibration bodies and coral colonies proved the accuracy of the surface area determination. Surface area quantifications derived from two different surface rendering techniques applied for scanning living coral colonies showed congruent results (mean deviation ranging from 1.32 to 2.03%). The validity of surface area measurement was verified by repeated measurements of the same coral colonies by three test persons. No significant differences between all test persons in all coral genera and in both surface rendering techniques were found (independent sample t-test: all n.s.). Data analysis of a single coral colony required approximately 15 to 30 min for a trained user using the isosurface technique regardless of the complexity and growth form of the latter, rendering the method presented in this study as a time-saving and accurate method to quantify surface areas in both living coral colonies and bare coral skeletons. Communicated by Biology Editor Dr Michael Lesser     

Encapsulation of tannase for the hydrolysis of tea tannins

Tannase was encapsulated in alginate, chitosan, carrageenan or pectin gel matrices, and in the case of alginate, coated with high or low molecular weight chitosan to reduce enzyme release. Cross-linking with glutaraldehyde also improved enzyme retention. Active enzyme preparations were obtained, although carrageenan gels were unstable in tea. Tannase activity was evaluated by reduction in centrifugable (flocculated) tea solids, and a reduction in tea cream measured turbidimetrically after removal of flocculated solids. Tannin interactions with the polysaccharide gels increased the level of centrifugable solids (flocculent) in the tea. An optimum bead formulation consisted of an alginate core, coated with chitosan and cross-linked with glutaraldehyde. Both core and coating materials contained active enzyme. Beads were prepared in a single step procedure involving extrusion of alginate/tannase solution into a hardening bath containing tannase-loaded, chitosan solution. Tannase retained hydrolytic activity through three successive batch cycles, for a total period of 39h processing, and tea cream was visibly removed by treatment with the immobilized tannase. Activity remained stable during 1-month bead storage under refrigeration.     

Process optimization for microencapsulation of probiotic yeasts

Background

Microencapsulation is a technique which improves the survival and viability of probiotics. We demonstrate encapsulation of five potential probiotic yeasts with alginate and gum as encapsulation matrices to improve their gastrointestinal transit.

Methods

Gum extracted from various cereals viz. rice, oats, barley, finger millet and pearl millet along with alginate have been used to encapsulate five potential probiotic yeasts. Screening was carried out by measuring swelling index, encapsulation efficiency and nutritional value of microcapsules encapsulated with alginate and gum. The concentration of OBG, sodium alginate and inoculum dosage of probiotic yeasts was optimized using response surface methodology (RSM). Efficiency of alginate OBG microcapsules with or without coating materials viz. whey protein and chitosan also tested. The mucoadhesion ability and storage stability of alginate OBG microcapsules with coating materials were tested.

Results

Highest encapsulation efficiency of probiotic yeasts was noted using oats bran gum (OBG) microcapsules along with alginate in all the five probiotic yeasts. Notably whey protein coated microcapsules showed maximum GIT tolerance (95%) and mucoadhesion (90%) for L. starkeyi VIT-MN03. The minimum loss of viability was observed in L. starkeyi VITMN03 microcapsules on 60th day of storage.

Conclusions

This is the first report on optimization and survival of microencapsulated probiotic yeasts under simulated GIT conditions using natural gum and alginate as encapsulation matrices and whey protein as coating material.

     

A Review of Methods for Measuring the Surface Area of Stream Substrates

Surface area measurement is a common component of benthic research, especially in the quantification of chlorophyll. Multiple techniques are available and 10 are described: artificial substrates, area-specific sampling, geometric approximation, stone shape equations, foil wrapping, grids, stamps, wetted layer, particle layer, and planar area measurement. A literature search of 130 papers indicated the most common methods: using artificial substrates of known area, subsampling a specific area using a template or sampler, measuring stone dimensions and using an equation to derive area, and using the weight of foil wrapped on stones. Methods were compared using spheres of known area, smooth and rough granite stones, and plastic macrophytes. Most methods produced highly correlated measurements and accurately estimated surface area. The wetted layer method was sensitive to stone roughness and plant complexity, but may overestimate the area of complex surfaces. Replication of one method by 10 biologists indicated that individual differences in technique can affect surface area values. Factors to consider in choosing an appropriate method include ease of use, characteristics of the substrates (e.g., porosity and flexibility), fineness of scale in measuring area, and whether methods must be field-based or can include laboratory techniques.     

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%     

Technical note: A new method for measuring long bone curvature using 3D landmarks and semi‐landmarks

Here we describe and evaluate a new method for quantifying long bone curvature using geometric morphometric and semi‐landmark analysis of the human femur. The technique is compared with traditional ways of measuring subtense and point of maximum curvature using either coordinate calipers or projection onto graph paper. Of the traditional methods the graph paper method is more reliable than using coordinate calipers. Measurement error is consistently lower for measuring point of maximum curvature than for measuring subtense. The results warrant caution when comparing data collected by the different traditional methods. Landmark data collection proves reliable and has a low measurement error. However, measurement error increases with the number of semi‐landmarks included in the analysis of curvature. Measurements of subtense can be estimated more reliably using 3D landmarks along the curve than using traditional techniques. We use equidistant semi‐landmarks to quantify the curve because sliding the semi‐landmarks masks the curvature signal. Principal components analysis of these equidistant semi‐landmarks provides the added benefit of describing the shape of the curve. These results are promising for functional and forensic analysis of long bone curvature in modern human populations and in the fossil record. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Volume scanning electron microscopy for imaging biological ultrastructure

Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre‐scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error‐prone manual tasks of cutting and handling large numbers of sections, and imaging them one‐by‐one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z‐resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block‐face electron microscopy (SBEM), focused ion beam SEM (FIB‐SEM) and automated tape‐collecting ultramicrotome SEM (ATUM‐SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research.     

Computerized histopathologic assessment of malignant potential. III. Refinements of measurement and data analysis

The nucleolar area of uveal melanomas, measured from standard hematoxylin-and-eosin-stained microslides , has been shown in previous work to correlate well with survival following enucleation of the tumor-bearing eye. Despite this correlation, the accuracy of the original system for measuring nucleolar area was affected by several sources of error: (1) the algorithm by which area was computed underestimated true area, (2) geometric and optical factors caused overestimation of nucleolar area unless measured in the center of the microscopic field of view and (3) the area of small nucleoli, as the result of several possible mechanisms, contains less useful information than the area of large nucleoli. This paper presents methods introduced to reduce error from these sources and demonstrates that the predictive value of nucleolar measurements is relatively insensitive to high levels of random variation.     

Geometrical Custom Modeling of Human Cornea In Vivo and Its Use for the Diagnosis of Corneal Ectasia

Aim

To establish a new procedure for 3D geometric reconstruction of the human cornea to obtain a solid model that represents a personalized and in vivo morphology of both the anterior and posterior corneal surfaces. This model is later analyzed to obtain geometric variables enabling the characterization of the corneal geometry and establishing a new clinical diagnostic criterion in order to distinguish between healthy corneas and corneas with keratoconus.

Method

The method for the geometric reconstruction of the cornea consists of the following steps: capture and preprocessing of the spatial point clouds provided by the Sirius topographer that represent both anterior and posterior corneal surfaces, reconstruction of the corneal geometric surfaces and generation of the solid model. Later, geometric variables are extracted from the model obtained and statistically analyzed to detect deformations of the cornea.

Results

The variables that achieved the best results in the diagnosis of keratoconus were anterior corneal surface area (ROC area: 0.847, p<0.000, std. error: 0.038, 95% CI: 0.777 to 0.925), posterior corneal surface area (ROC area: 0.807, p<0.000, std. error: 0.042, 95% CI: 0,726 to 0,889), anterior apex deviation (ROC area: 0.735, p<0.000, std. error: 0.053, 95% CI: 0.630 to 0.840) and posterior apex deviation (ROC area: 0.891, p<0.000, std. error: 0.039, 95% CI: 0.8146 to 0.9672).

Conclusion

Geometric modeling enables accurate characterization of the human cornea. Also, from a clinical point of view, the procedure described has established a new approach for the study of eye-related diseases.     

人类股骨断面面积与形状的不对称性——基于三维激光扫描的形态测量分析

人类股骨横断面面积、形状及其左右侧差异记载的人类演化、人群差异及生存活动的重要信息一直为古人类学研究所关注。多年来, 对股骨断面的研究通常采用破坏性地切割或者制作模型的方法。本文利用三维激光表面扫描技术, 无损、快捷、方便地获取了20对现代中国人左右侧股骨外轮廓的三维数据, 采用CAD软件及几何形态测量方法对两侧股骨断面轮廓的大小及形状进行了对比和分析。初步研究结果发现: 两侧股骨的横断面相对面积差异极其显著, 绝对面积差异不显著, 不对称方式表现为波动不对称性, 而不是偏向不对称性; 个体之间两侧股骨横断面外轮廓形状的波动不对称性极其显著, 偏向不对称性虽有差异但不显著; 平均形状和面积分析结果似乎表明股骨稍有偏左侧优势。虽然本文所采用的标本量有限, 所得出的结论需要更多标本的进一步验证, 但是, 本文的研究结果提示利用三维激光扫描技术获取股骨横断面外轮廓数据, 并采用形态测量方法分析确实能够揭示出一些以往研究方法不能发现的重要信息, 这种研究骨骼不对称性的新方法值得进一步的应用。     

Surface area–volume ratios in insects

Body mass, volume and surface area are important for many aspects of the physiology and performance of species. Whereas body mass scaling received a lot of attention in the literature, surface areas of animals have not been measured explicitly in this context. We quantified surface area–volume (SA/V) ratios for the first time using 3D surface models based on a structured light scanning method for 126 species of pollinating insects from 4 orders (Diptera, Hymenoptera, Lepidoptera, and Coleoptera). Water loss of 67 species was measured gravimetrically at very dry conditions for 2 h at 15 and 30 °C to demonstrate the applicability of the new 3D surface measurements and relevance for predicting the performance of insects. Quantified SA/V ratios significantly explained the variation in water loss across species, both directly or after accounting for isometric scaling (residuals of the SA/V ～ mass^2/3 relationship). Small insects with a proportionally larger surface area had the highest water loss rates. Surface scans of insects to quantify allometric SA/V ratios thus provide a promising method to predict physiological responses, improving the potential of body mass isometry alone that assume geometric similarity.     

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

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

Comparison of several digital and stereological methods for estimating surface area and volume of cells studied by confocal microscopy.

BACKGROUND: The implementation of different methods for estimating the surface area and volume of cells studied by confocal microscopy was developed. The methods were compared from the point of view of their precision, applicability and efficiency. METHODS: Interactive stereological methods (spatial grid method, fakir method, Cavalieri principle) as well as automatic digital methods (digital Crofton method, voxel counting, triangulation method, iso-intensity contouring method) were considered. The methods were tested on model geometrical solids and on real volume images consisting of a stack of serial sections encompassing entire tobacco BY-2 cells or cell chains. RESULTS: It is shown that many of the studied methods are very precise when applied to cells of simple or moderately complex shapes. The automatic digital methods are fast and precise but their applicability is limited by the necessity to segment automatically the object surface and to find an optimal resolution. This limitation is not present in stereological methods which are applied interactively and thus are more time-consuming. CONCLUSIONS: The presented implementations of the fakir method and the Cavalieri principle enable interactive, unbiased and efficient estimation of the cell surface area and volume. The recommended steps for measuring the surface area and/or volume of objects studied by confocal microscopy are described.     

Indirect assessment of callus fresh weight by non-destructive methods

The size of callus of Nicotiana plumbaginifolia was measured by determinations of fresh weight (FW), area (electronic planimeter and a point-counting method) and width (standard width and greatest width). All these methods, with the exception of the standard width measurements, were found to produce adequate substitutes for fresh weight.Particular advantages apply to the use of the point-counting method, but the relationship between callus area and point interval was found to be critically important in determining the accuracy of measurements. The use of surface dimensions rather than FW permits continuous measurement of callus size without disturbance of the callus or its environment within the containers.Abbreviation FW fresh weight