Digital Image Analysis Techniques Research Articles

Strain Localization in Combined Axial-Torsional Testing on Kaolin Clay

A series of combined axial-torsional tests were performed to study the 3D mechanical behavior of kaolin clay in an undrained condition. Using the digital image analysis technique, discrete local deformation of the surface of a hollow cylindrical specimen under loading was recorded. A linear interpolation method was used to generate a continuous deformation and strain field of the specimen based on the recorded discrete local deformations. Evolution of shear band was vividly visualized and recorded during the loading process for various inclinations of major principal stress. The theory of strain localization on continuous bifurcation was briefly reviewed and applied to the Mohr-Coulomb model, and a single hardening model incorporating the concept of loading-history-dependent plastic potential was developed by the writers. The largest critical plastic modulus and orientation of the shear bands were predicted by using the theoretical solution. Significant disagreement was observed between the experimental results and theoretical predictions related to the initial occurrence of strain localization and the inclination of fully developed shear bands.

A 24-Hour Dental Plaque Prevention Study with a Stannous Fluoride Dentifrice Containing Hexametaphosphate

Recently, a novel antibacterial fluoride dentifrice containing stannous fluoride and sodium hexametaphosphate (CrestPRO-HEALTH) was introduced. A digital plaque image analysis (DPIA) technique was used to quantify in situ plaque formation in a population carrying out a phased intervention protocol that included: (1) an initial treatment regimen including toothbrushing with standard sodium fluoride dentifrice in conventional bid brushing, (2) a second treatment regimen where a modified hygiene regimen was applied using standard sodium fluoride dentifrice including a period of 24 hours of non-brushing, and (3) a third treatment regimen where the 24-hour non-brushing regimen was continued using the antimicrobial stannous fluoride/sodium hexametaphosphate dentifrice. The quantitative evaluation of plaque formation was assessed in morning measurements following either standard evening hygiene (treatment period 1) or 24 hours since brushing (treatment periods 2 and 3). Post-brushing plaque measurements were also taken in each treatment regimen. Sixteen subjects completed all three treatment regimens with no side effects or oral complaints. Morning plaque coverage in treatment period 1 was 13.3%. Plaque coverage significantly increased in treatment period 2 when pre-bedtime brushing was discontinued, with 24-hour growth covering 18.4% of the dentition. Intervention of the antimicrobial stannous fluoride/hexametaphosphate dentifrice in treatment period 3 provided significant inhibition of plaque regrowth over 24 hours (15.2% coverage, a 17% reduction vs. sodium fluoride dentifrice control). These results support the strong retention and lasting antimicrobial efficacy of high stabilized stannous fluoride/sodium hexametaphosphate dentifrices.

Characterization of biofilm structure and its effect on membrane permeability in MBR for dye wastewater treatment

Two membrane bioreactors were operated at aerobic (DO=6.0 mg/L) and anoxic (DO<0.3 mg/L) conditions for the treatment of synthetic dye wastewater to determine the effect of dissolved oxygen on membrane filterability. The rate of membrane fouling for the anoxic MBR was five times faster than that for the aerobic MBR. Differences in the nature of the biofilm that was formed on the membrane surface as the result of different DO level was the main factor in the different fouling rates. The biofilm structure was characterized using digital image analysis techniques. Biofilm images were obtained using confocal laser scanning microscopy (CLSM) at various operation points. Structural parameters were then computed from these images using an image analysis software (ISA-2). The structural parameters indicated that the anoxic biofilm was thinner than the aerobic biofilm but the anoxic biofilm was spread out on the membrane surface more uniformly and densely, resulting in the higher membrane fouling. Based on the extracellular polymeric substances (EPS) visualization and quantification, it was also found that EPS, key membrane foulants were spread out more uniformly in the anoxic biofilm in spite of lower amount of EPS compared to that in the aerobic biofilm.

Influence of alcohol and tobacco habits on peri‐implant marginal bone loss: a prospective study

A prospective clinical study was conducted to explore the possible link between peri-implant bone loss and the widespread habits of tobacco smoking and alcohol consumption. One hundred and eighty-five patients who received 514 implants were followed up for 3 years. Peri-implant marginal bone loss was evaluated by digital panoramic radiography and image analysis techniques. Multivariate analysis showed that peri-implant marginal bone loss was significantly related to a daily consumption of >10 g of alcohol, tobacco use and increased plaque levels and gingival inflammation. The present results indicate that daily alcohol consumption and tobacco use may have a negative influence on predictable long-term implant treatment outcomes, producing peri-implant bone loss and compromising restorative treatment with implant-supported prostheses.

Droplet Evaporation Study Applied to DNA Chip Manufacturing

DNA chips are potentially powerful technologies for genotyping and gene expression profiling that rely on comparative analyses of up to thousands of "spots of analysis" on a glass support. The spot quality throughout the support influences spot-to-spot variations within an array and the repeatability of data across experiments. For glass slide DNA microarrays, droplets of DNA solution are deposited on functionalized glass slides and left to react through complete evaporation of the droplet. On hydrophobic flat surfaces, different modes of droplet evaporation can be attained. Under atmospheric pressure, water droplets tend to evaporate under two main regimes. Initially, the droplet flattens with a constant contact area, and then the droplet shrinks at a constant contact angle. As a result, the diameter and morphology of thousands of spots on microarrays are not uniform. This leads to poor and unreliable data processing results. In this work, we report the evaporation of an aqueous solution under a constant contact area mode. Evaporation under reduced pressure and the effect of reagent additives to the solution have been investigated. Video microscopy and digital image analysis techniques were applied to monitor the evaporation of the droplets. A mixture of surfactants was developed to maintain a constant area regime during evaporation and to form homogeneous spots. The control of some physicochemical properties (wetting, evaporation rate) of the droplet allows the formation of well-controlled spots compatible with DNA grafting. The influence of surfactant molecules on the mechanisms of evaporation is also discussed.

Critical testing of Earth's oldest putative fossil assemblage from the ∼3.5 Ga Apex chert, Chinaman Creek, Western Australia

Structures resembling cyanobacterial microfossils from the ca. 3465 Ma old Apex chert of the Warrawoona Group in Western Australia have until recently been accepted as providing the oldest morphological evidence for life on Earth, and have been taken to support an early beginning for oxygen-releasing photosynthesis. Eleven species of filamentous prokaryote, principally distinguished by shape and geometry, have been put forward as meeting the criteria required of authentic Archaean microfossils. They were contrasted with other microfossils that were dismissed as either unreliable or irreproducible. The aim of this paper is to provide a detailed account of research recently reported by us on the type and recollected material, involving optical and electron microscopy, digital image analysis and other techniques. All previously figured holotype materials are illustrated here, and the context for all the published materials is re-evaluated. The Apex chert ‘microfossils’ occur near the top of a 1.5-km long chert dyke complex associated with major synsedimentary growth faults. Highly localised, glassy felsic tuffs erupted explosively from this and other fissures during the early stages of volcanism, and were followed by the deposition of essentially hydrothermal black and white BaSO 4 rich cherts that infiltrated the feeder dykes, underplating and dilating adjacent stratiform cherts before the start of the next volcanic cycle. The Apex chert ‘microfossils’ occur within multiple generations of these metalliferous hydrothermal vein cherts some 100 m down the dyke system. Comparable structures occur in associated volcanic vent glass and in hydrothermal cherts at least 1 km deep. We find no supporting evidence for a primary biological origin. We reinterpret the purported microfossil-like structures as pseudofossils that formed from the reorganization of carbonaceous matter, mainly during recrystallization from amorphous to spherulitic silica.

The application of digital photogrammetry and image analysis techniques to derive tree and stand characteristics

Ground-based forest inventory surveys can provide highly accurate measurements of tree and stand characteristics, but these are expensive to carry out. Aerial photography has been used for several decades as a tool in forest management and inventory. However, conventional methods of interpretation are both time-consuming and costly, with results varying among interpreters. With continuing development of personal computer technology, aerial photographs have become more accessible for digital analysis. This paper presents the potential operational use of digitized aerial photographs for the estimation of tree and stand characteristics of two forest plantations of Sitka spruce (Picea sitchensis (Bong.) Carrière) in Scotland. The digitized aerial photographs were processed using softcopy photogrammetry, and image analysis techniques were used for individual tree crown delineation. For the first site the estimations of stand top height, basal area, volume, biomass, and density (–23.7%) were similar to the ground-measured stand characteristics (±10%), whereas for the second site the estimations were less accurate mainly because of the nonoptimal illumination conditions during the acquisition of the aerial photographs.

Compatibility of the green fluorescent protein and a general nucleic acid stain for quantitative description of a Pseudomonas putida biofilm

Better understanding of biofilm development is essential for making optimal use of beneficial biofilms as well as for devising effective control strategies for detrimental biofilms. Analysis of biofilm structure and quantification of biofilm parameters using optical (including confocal) microscopy and digital image analysis techniques are becoming routine in many laboratories. The purpose of this study was to evaluate a dual labeling technique based on fluorescence signals from the green fluorescent protein (GFP) and those resulting from staining with the general nucleic acid stain SYTO 60 for the quantitative description of a model biofilm. For this purpose, a Pseudomonas putida KT2442 derivative was genetically tagged with the green fluorescent protein gene. Biofilm formation by this strain was investigated using flow cells and confocal laser scanning microscopy (CLSM). Percentage surface coverage as well as microcolony size quantified using GFP and SYTO 60 signals showed significant correlation ( R=0.99). The results indicated that intrinsic labelling of this model biofilm using constitutively expressed proteins such as GFP can be used for real-time biofilm observation and generation of reliable quantitative data, comparable to those obtained using conventional methods such as nucleic acid staining. Non-destructive time series observation of GFP-expressing biofilms in flow-cells can thus be confidently used for four-dimensional ( x, y, z, t) analysis and quantification of biofilm development. The results also point to the possibility of using GFP and SYTO 60 to study dual species biofilms, as quantitative data generated using both fluorophore signals are comparable.

Measuring Cartilage Morphology with Quantitative Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) is a three-dimensional imaging technique with unparalleled ability to delineate articular cartilage morphology in health and disease. In this article we will review work on the assessment of cartilage morphology with quantitative magnetic resonance imaging and its relevance to the study of cartilage anatomy, physiology, deformation, disease status, disease progression, and response to treatment. The review outlines available pulse sequences and techniques for segmentation and morphological analysis of cartilage morphology. It addresses the accuracy (validity) and precision (reproducibility) of these techniques and summarizes studies on cartilage deformation in intact joints. This article will also review work on determinants and functional adaptation of cartilage morphology and describe changes seen in osteoarthritis. We conclude that fat-suppressed or water excitation gradient-echo magnetic resonance sequences and state-of-the-art digital image analysis techniques display high accuracy and adequate precision for quantitative assessment of cartilage morphology. This renders these techniques powerful and promising tools for cartilage and osteoarthritis research.

Automatic Tracking, Feature Extraction and Classification of C. elegans Phenotypes

This paper presents a method for automatic tracking of the head, tail, and entire body movement of the nematode Caenorhabditis elegans (C. elegans) using computer vision and digital image analysis techniques. The characteristics of the worm's movement, posture and texture information were extracted from a 5-min image sequence. A Random Forests classifier was then used to identify the worm type, and the features that best describe the data. A total of 1597 individual worm video sequences, representing wild type and 15 different mutant types, were analyzed. The average correct classification ratio, measured by out-of-bag (OOB) error rate, was 90.9%. The features that have most discrimination ability were also studied. The algorithm developed will be an essential part of a completely automated C. elegans tracking and identification system.

The Effects of Plant Populations on Pickling Cucumber Canopy Dynamics and Yield

Michigan is the national leader for pickling cucumber production. However, over the last few years growers have witnessed a considerable decline in marketable yield, mainly attributed to fruit rot caused by Phytophthora capsici. Phytophtora develops rapidly under high relative humidity, a situation commonly found with narrow rows. Growers are interested in using wider rows but would like to know if there are any associated yield reductions. This study was conducted in 2003 to measure the effects of cucumber plant populations on canopy dynamics and fruit yield. Cucumbers were grown with between-row spacing of 30.5, 45.7, 61.0, and 76.2 cm, and in-row spacing of 10.2, 12.7, and 15.2 cm. A split-plot design with four replications was used. Row spacing was the main plot factor, and in-row spacing the subplot factor. Soil covered by plant canopy was monitored throughout the growing season using digital image analysis techniques. At harvest, the number of fruits per plant and marketable yield for the different grades were measured. Cucumber canopy remained open during the major part of the growing season when wide rows (61.0 and 76.2 cm) were used. The number of fruits per plant increased from an average of 1.5 fruits at 30.5 cm to 2.0 fruits per plant at 61.0 cm. Further widening of row spacing to 76.2 cm slightly reduced the number of fruits per plant. Therefore, the optimum row spacing would be 61.0 cm if the number of fruits per plant was the only parameter being measured. Cucumber marketable yield was similar with 30.5, 45.7, and 61.0 cm spacing between the rows. With 76.2-cm rows, yield reduced slightly. These results suggest that cucumber plant density can be reduced substantial with limited yield penalty.

The 3D structure of fabric and its relationship to liquid and vapor transport

Polymeric carrier fabrics are commonly used in many industrial processes including manufacture of paper and board. Apart from acting as a carrier for the compressible porous material during the manufacturing process, the synthetic woven fabrics comprising mainly of poly ethylene terypthalate (PET) yarns, impart valuable product attributes, i.e. softness, bulk, absorbency, etc. in consumer products. The three-dimensional structure of the fabrics plays a critical role in deciding the manufacturing and energy efficiency as well as product end-use properties. X-ray micro computed tomography (X-μCT) provides a non-intrusive technique to visualize and analyze the three-dimensional structure of porous materials such as paper [The 3 Dimensional Structure of Paper and Its Relationship to Liquid and Vapor Transport, The Science of Papermaking, p. 1289; Tappi J 84 (2001) 1; APPITA 55 (2002) 230]. In this paper, we use this technique to visualize the three-dimensional structure of polymeric fabrics commonly used in paper manufacture [The 3 Dimensional Structure of Paper and Its Relationship to Liquid and Vapor Transport, The Science of Papermaking, p. 1289; Tappi J. 84 (2001) 1; APPITA 55 (2002) 230]. Digital image analysis techniques based on mathematical morphology and stereology were used to determine traditional pore descriptors such as porosity, yarn–void interfacial area, tortuosity and hydraulic radii distribution in the two principal orthogonal directions [The 3 Dimensional Structure of Paper and Its Relationship to Liquid and Vapor Transport, The Science of Papermaking, p. 1289; APPITA 55 (2002) 230]. Comparison of the average yarn diameter by X-μCT and image analysis and physical measurement using light microscopy agreed to within 3% indicating the good accuracy of the X-ray technique. The differences in fabric pore structural characteristics between the in-plane and transverse directions reported here help explain the differences in liquid and vapor transport in the two principal directions. Lattice-Boltzmann simulations of fluid flow and Monte-Carlo simulations of vapor diffusion through actual 3D structures of fabric provide a direct method to predict the permeability and diffusivity characteristics of these complex media. Comparison of structural characteristics between image analysis and simulations show reasonable agreement.

Two‐Phase Flow Characterization by Automated Digital Image Analysis. Part 2: Application of PDIA for Sizing Sprays

AbstractA series of experiments were conducted in order to assess the robustness and accuracy of a recently developed digital image analysis technique (PDIA). This paper investigates the application of the PDIA technique to the sizing of relatively small fuel droplets of diameters in the range 5 to 30 μm produced by a pressure‐swirl atomizer. The measurement performance of the PDIA system has been assessed in terms of individual object diameters and also number and volume probability density functions of diameter in comparison to phase Doppler anemometry (PDA) data obtained under identical conditions. PDIA measurements revealed good agreement with spray data obtained by PDA at a measurement location 36 diameters downstream from the nozzle orifice with differences in the arithmetic mean diameter, D10 and volume mean diameter, D30 of approximately 5 and 3% respectively. The PDIA technique was shown to detect the presence of very large, predominantly non‐spherical droplets whose diameters were in excess of 100 μm. These droplets, although few in number constitute a significant proportion of the total spray volume and would have otherwise been either erroneously measured or have passed through the probe volume undetected using PDA due to non‐sphericity. Smaller objects may also be measured correctly by both methods although sensitivity to signal‐to‐noise ratio, for both methods can generate spurious and contradictory errors.

Hydrodynamic modelling of dense gas-fluidised beds: comparison and validation of 3D discrete particle and continuum models

A critical comparison of a hard-sphere discrete particle model, a two-fluid model with kinetic theory closure equations and experiments performed in a pseudo-two-dimensional gas-fluidised bed is made. Bubble patterns, time-averaged particle distributions and bed expansion dynamics measured with a nonintrusive digital image analysis technique are compared to simulation results obtained at three different fluidisation velocities. For both CFD models, the simulated flow fields and granular temperature profiles are compared. The effects of grid refinement, particle–wall interaction, long-term particle contacts, particle rotation and gas–particle drag are studied. The mechanical energy balance for the suspended particles is introduced, and the energy household for both CFD models is compared. The most critical comparison between experiments and model results is given by analysis of the bed expansion dynamics. Though both models predict the right fluidisation regime and trends in bubble sizes and bed expansion, the predicted bed expansion dynamics differ significantly from the experimental results. Alternative gas–particle drag models result in significantly different bed dynamics, but the gap between model and experimental results cannot be closed. In comparison with the experimental results, the discrete particle model gives superior resemblance. The main difference between both CFD models is caused by the neglect of particle rotation in the kinetic theory closure equations embedded in the two-fluid model. Energy balance analysis demonstrates that over 80% of the total energy is dissipated by sliding friction. Introduction of an effective restitution coefficient that incorporates the additional dissipation due to frictional interactions significantly improves the agreement between both models.

Microstructure-dependent densification of polar firn derived from X-ray microtomography

AbstractThe densification of dry polar snow and firn results in a continuous increase of density with depth accompanied by significant density fluctuations within seasonal layers. Density measurements of high spatial resolution reveal a persistent minimum of density fluctuations in the vicinity of the snow–firn transition (0.55–0.65 g cm-3) in firn-core records. In this study we give an explanation for the fluctuation minimum by applying a new method of X-ray microtomography to obtain three-dimensional (3-D) structural data of a Greenland firn core. At 13 different depths between 10 and 78 m a set of 16 samples of 40 cm total length for each depth interval was measured. A reconstructed firn segment of 40 cm covers 1–2 years of snow accumulation. Using digital image analysis techniques, different structural parameters are estimated including 3-D pore and particle sizes and specific surface areas. It is shown that the densification rates of snow and firn layers consisting of coarse particles are much higher than those consisting of fine particles within the same depth interval. This causes a density crossing of fine- and coarse-grained layers with a minimum of density variations at the crossover point. This crossing-over implies that formerly dense layers in the seasonal density signal are not of the same origin as dense layers in the deeper part of the firn column and that the seasonal density signal will totally change shape with depth. It is speculated that in coarse- and fine-grained firn the dominant mechanism of densification acts over different regimes of density.

Two‐Phase Flow Characterization by Automated Digital Image Analysis. Part 1: Fundamental Principles and Calibration of the Technique

AbstractA recently developed digital image analysis technique potentially capable of sizing particles of arbitrary shape and size and with wide dynamic range is examined. Shadow images of droplets or particles are produced in this case by back‐illumination using an infra‐red diode laser. The measurement performance of the particle/droplet image analysis (PDIA) system has been assessed initially in terms of individual object diameters and the aim of this experimental investigation was to assess the robustness and accuracy of the technique. The first part of this paper provides a description of the fundamental principles of the technique followed by a thorough description of the calibration procedure in which the image processing routine was verified with calibration data of known particle sizes. The lens‐camera optical behavior has been characterized whilst the relative uncertainties of various parameters such as the depth‐of‐field dependence on particle diameter, threshold level sensitivity and image signal‐to‐noise issues have been quantified and their effects on measurement accuracy discussed. Calibration data revealed that the depth‐of‐field varied with object diameter approximately linearly in the measured range 18 to 145 μm. The calibration data is subsequently incorporated into the PDIA processing algorithm and for defocused droplets or particles, enables their true diameter to be estimated by analyzing the droplet image properties. Greater emphasis on the application of the PDIA technique is deferred to the second part of this two‐part paper where droplet size data obtained from a hollow‐cone spray is compared with similar data measured via phase Doppler anemometry (PDA).

Abundance estimation of rocky shore invertebrates at small spatial scale by high-resolution digital photography and digital image analysis

We have tested both the usefulness of high-resolution digital photography for data acquisition and digital image analysis, by non-supervised classification and high pass filter, for recognition and abundance estimation of benthic intertidal organisms. These digital tools were compared with visual scan and photo quadrat conventional methods. The comparison was done using 40 quadrats (10×5 cm) randomly selected along a 5-m transect on the rocky shore of the Pemaquid Point, Maine, USA. ANOVA for repeated measures was used to test differences among methods. Monte Carlo simulation analysis was used to explore differences among methods over a large set of data ( n=100, 500, 1000 quadrats). Differences among methods were observed when 40 quadrats were used. Tukey multiple comparison test showed that abundance estimation from visual scan, photo quadrat and digital image analysis by high pass filter do not differ significantly among them but differ from non-supervised classification results. Due to its accurate estimation, high pass filter (Prewitt) method was chosen as the most reliable digital method to estimate species abundance. Monte Carlo simulation of visual scan, photo quadrat and high pass filter results showed significant differences when the number of quadrats was larger. These results showed that the combined use of digital photography and digital image analysis techniques for the acquisition and analysis of recorded data is a powerful method for the study of intertidal benthic organisms. Results produced using these techniques were similar than those produced by conventional methods but were obtained in a much-reduced time.

Digital image analysis measurements of bed expansion and segregation dynamics in dense gas-fluidised beds

One of the most crucial steps in the development of fundamental hydrodynamic models is the validation of these models with accurate, detailed experimental data. Therefore a whole-field, non-intrusive digital image analysis technique has been developed which enables measurement of bed expansion and segregation dynamics of coloured particles in dense gas-fluidised beds. The development, calibration and accuracy of the technique are discussed in detail. The image analysis technique traces bubbles and voidage waves accurately, whereas the mixture composition in a fluidised bed could be determined within 10%.Experiments have been carried out with 1.5 and 2.5 mm coloured glass beads, for which particle–particle and particle–wall collision parameters were accurately known. They were performed in pseudo two-dimensional laboratory scale fluidised beds with a simple rectangular geometry and well-defined gas inflow conditions. An extensive set of results obtained with both mono-disperse systems and binary mixtures, suitable for validation of fundamental hydrodynamic models, is presented.

Tolerance of Mycelium of Different Genotypes of Phytophthora infestans to Freezing Temperatures for Extended Periods

ABSTRACT Mycelium of Phytophthora infestans, the causal agent of potato late blight, can initiate crop infections over successive years by overwintering in infected potato tubers that survive as seed in fields or within cull piles. This study used four different genotypes of P. infestans to evaluate the influence of freezing temperatures on survival of mycelium in vitro. Sporangium-free mycelium of P. infestans US1, US8, US11, and US14 growing on rye agar plates was exposed to temperatures ranging from -20 and 0 degrees C (experiment A) for different periods up to 24 h and from -5 and 0 degrees C (experiment B) for periods up to 5 days. Cultures were incubated at 12 degrees C after exposure, and survival of the cultures was estimated after 28 days by a digital image analysis technique that measured the average reflectance intensity (ARI) of images of the mycelium of temperature-treated cultures. The ARI values of treated cultures were compared with the growth of mycelium in negative controls (mycelium not present) and positive controls (mycelium exposed to 12 degrees C for an equivalent period), and determination of recovery was based on statistical differences from the controls. There were significant differences in ARI values among genotypes, temperature treatment, and exposure periods in all experiments. An index of recovery was calculated for each genotype at all treatment temperatures and exposure periods for both experiments. In experiment A, exposure of mycelium of P. infestans (all genotypes) to -20 and -10 degrees C proved lethal for exposure periods of more than 1 h. All genotypes showed some degree of recovery up to 24-h exposure at -5 and -3 degrees C. In both experiments, exposure of mycelium of P. infestans to 0 degrees C was not lethal to any genotype tested for any exposure period. In experiment B, all of the genotypes survived exposure up to 3 days at -3 degrees C to some degree, but at -5 degrees C, exposure of 1 day was lethal to all genotypes. Tolerance of freezing temperatures by mycelium of P. infestans may be an ecologically important survival mechanism and the increased tolerance of US8 and US14 may explain their predominance in cooler climates such as north-central United States.

Digital Image Analysis of Internal Light Spots of Appressoria ofColletotrichum acutatum

ABSTRACT The initial penetration process of appressoria of Colletotrichum acutatum on almond leaves was studied using digital image analysis of light micrographs and scanning electron microscopy. For image analysis, a series of sequential, partially focused digital micrographs of appressoria was analyzed to generate a single, completely focused montage image with a continuous in-focus depth of field. In studies on the development of the internal light spot (ILS), we observed that 50.4% of the appressoria formed an ILS after leaves were inoculated and incubated for 12 h at 20 degrees C, and that this increased to 95.8% after 24 h. Comparative image analyses of appressoria with and without ILSs using depth relief mapping and line profile software options showed that the ILS had a depth relief that was below that of the leaf surface. Depth relief analysis in the ILS region during incubation revealed an increase in depth in this area of up to 1.8 mum in some of the appressoria. A comparative morphological study of the ILS in montage images and the penetration pore of appressoria in scanning electron micrographs showed similar shapes and dimensions of the two structures in the appressorium. Light micrographs of histological sections confirmed fungal penetration and internal vesicle formation in almond leaves within 36 h after inoculation and incubation at 20 degrees C. This study represents the first direct evidence that the ILS in appressoria corresponds to the penetration pore and the developing penetration peg using a rapid, digital image analysis technique.