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PubMed1994

Analysis of changes in proteoglycan content in murine articular cartilage using image analysis.

The extracellular matrix of articular cartilage consists mainly of type II collagen and large aggregating proteoglycan (aggrecan). During arthritis and other joint diseases, the proteoglycan (PG) level of cartilage matrix is diminished, leading to impairment of normal joint function. A new method is described for measuring the changes in PG content of murine articular cartilage. The method is based on the automated densitometric analysis of patellar cartilage of standard, safranin O-stained sections of whole murine knee joints. It appeared to be possible to measure optical density in parallel layers of articular cartilage with high reproducibility. Approximately 25 sections can be evaluated within 1 h. Measuring a single section 10 times resulted in a coefficient of variation (CV) of 0.1-1.4%. A mean CV of 5-14% was calculated when a group of 18 sections was analyzed in quintuplicate. To validate the procedure, changes in PG content induced by arthritis or by intra-articular injection of TGFbeta-1 were analyzed by the image analysis method, the dimethylmethylene blue (DMB) assay and by visual grading. Although not a quantitive method, the newly developed image analysis method appeared to be more sensitive in detecting significant change in PG content of murine articular cartilage than the DMB method or visual grading. The image analysis method makes it possible to measure changes in PG content of specific areas of articular cartilage with higher sensitivity than the DMB method and eliminating the bias inherent to visual grading by human observers.

Animals↗

PubMed1996

Methods in quantitative image analysis.

The main steps of image analysis are image capturing, image storage (compression), correcting imaging defects (e.g. non-uniform illumination, electronic-noise, glare effect), image enhancement, segmentation of objects in the image and image measurements. Digitisation is made by a camera. The most modern types include a frame-grabber, converting the analog-to-digital signal into digital (numerical) information. The numerical information consists of the grey values describing the brightness of every point within the image, named a pixel. The information is stored in bits. Eight bits are summarised in one byte. Therefore, grey values can have a value between 0 and 256 (2(8)). The human eye seems to be quite content with a display of 5-bit images (corresponding to 64 different grey values). In a digitised image, the pixel grey values can vary within regions that are uniform in the original scene: the image is noisy. The noise is mainly manifested in the background of the image. For an optimal discrimination between different objects or features in an image, uniformity of illumination in the whole image is required. These defects can be minimised by shading correction [subtraction of a background (white) image from the original image, pixel per pixel, or division of the original image by the background image]. The brightness of an image represented by its grey values can be analysed for every single pixel or for a group of pixels. The most frequently used pixel-based image descriptors are optical density, integrated optical density, the histogram of the grey values, mean grey value and entropy. The distribution of the grey values existing within an image is one of the most important characteristics of the image. However, the histogram gives no information about the texture of the image. The simplest way to improve the contrast of an image is to expand the brightness scale by spreading the histogram out to the full available range. Rules for transforming the grey value histogram of an existing image (input image) into a new grey value histogram (output image) are most quickly handled by a look-up table (LUT). The histogram of an image can be influenced by gain, offset and gamma of the camera. Gain defines the voltage range, offset defines the reference voltage and gamma the slope of the regression line between the light intensity and the voltage of the camera. A very important descriptor of neighbourhood relations in an image is the co-occurrence matrix. The distance between the pixels (original pixel and its neighbouring pixel) can influence the various parameters calculated from the co-occurrence matrix. The main goals of image enhancement are elimination of surface roughness in an image (smoothing), correction of defects (e.g. noise), extraction of edges, identification of points, strengthening texture elements and improving contrast. In enhancement, two types of operations can be distinguished: pixel-based (point operations) and neighbourhood-based (matrix operations). The most important pixel-based operations are linear stretching of grey values, application of pre-stored LUTs and histogram equalisation. The neighbourhood-based operations work with so-called filters. These are organising elements with an original or initial point in their centre. Filters can be used to accentuate or to suppress specific structures within the image. Filters can work either in the spatial or in the frequency domain. The method used for analysing alterations of grey value intensities in the frequency domain is the Hartley transform. Filter operations in the spatial domain can be based on averaging or ranking the grey values occurring in the organising element. The most important filters, which are usually applied, are the Gaussian filter and the Laplace filter (both averaging filters), and the median filter, the top hat filter and the range operator (all ranking filters). Segmentation of objects is traditionally based on threshold grey values. (AB

Algorithms↗

PubMed2003

Validation of an image analysis method for estimating coating thickness on pellets.

A digital image analysis method for the estimation of mean pellet size and coating thickness employing optical microscopy was evaluated. The coating thickness was expressed as the difference in mean projected area radius of the uncoated and the coated pellets. The repeatability, the intermediate precision and the robustness of the image analysis method were investigated. The repeatability and the intermediate precision of the image analysis method was excellent with a low degree of scatter between the measurements. The robustness investigation on the image analysis method illustrated the importance of controlling and monitoring the illumination technique utilised. Calibration of the image analysis equipment was of the highest importance. Using pellets with a high degree of sphericity and narrow size distribution, it was sufficient to use 1000 pellets to estimate the mean pellet size and the coating thickness with an accuracy of +/-1.2 microm. An equation is presented for an approximation of the number of pellets necessary to achieve a given accuracy in the estimation of mean pellet size and coating thickness.

Drug Implants↗

PubMed2003

Imaging of L-glutamate fluxes in mouse brain slices based on an enzyme-based membrane combined with a difference-image analysis.

A time-resolved imaging method for visualizing L-glutamate release in mammalian brain slices is proposed by using an enzyme membrane combined with a difference-image analysis. The enzyme membrane is composed of L-glutamate oxidase and horseradish peroxidase incorporated into a bovine serum albumin matrix. L-Glutamate triggers an enzyme-coupling reaction to convert a redox substrate (DA-64) to Bindschedler's Green, which gives a green color signal. The difference-image analysis is based on calculating slopes of a signal versus time (t) plot in the time range from (t - 40 s) to (t + 40 s) for visualizing L-glutamate release in terms of its flux (in mol min(-1) cm(-2)). The method was applied to a time-resolved imaging of hippocampal distribution of ischemia-induced L-glutamate release in mouse brain slices. The image of L-glutamate distribution showed that the level and time courses of L-glutamate fluxes were neuronal region-dependent. The maximum flux of L-glutamate at CA1 was observed at 7.7 min after ischemia. The flux at 7.7 min increased in the order of CA1 approximately CA3 > DG. The time course of the L-glutamate flux in the CA1 region was biphasic and that in the DG region was modestly biphasic. In the CA3 region, such biphasic release of L-glutamate was not seen. The ischemia-induced L-glutamate flux was accelerated when Mg2+ was omitted from an extracellular solution. The present enzyme membrane-based approach provides a useful method for visualizing distribution of L-glutamate release in the brain slices during ischemia.

Amino Acid Oxidoreductases↗

PubMed2006

Stereotactic statistical imaging analysis of the brain using the easy Z-score imaging system for sharing a normal database.

Statistical brain imaging analysis has good objectivity and reproducibility. In Japan, statistical parametric mapping (SPM) and three-dimensional stereotactic surface projections (3D-SSP) are used nationwide as statistical imaging analysis with standard brain coordinates. They often help to interpret brain single photon emission computed tomography (SPECT) images by avoiding possible pitfalls (e.g., effects of aging, atrophy) with which clinicians are unfamiliar. However, this type of analysis presents a problem: statistical processing requires many normal subject images. The easy Z-score imaging system (eZIS) is one of the statistical analysis methods that uses SPM processing in normalization and smoothing, and it has the function of image conversion leading to statistical analysis without a control database. Therefore, statistical analysis can be used in clinical practice by sharing a prepared normal database. By unifying the image quality by processing a shared database, this program has great potential for sharing patient imaging data in many hospitals. It is expected that the eZIS will help perform detailed analysis in many functional diseases in collaborative studies. This article describes the interpretation of brain SPECT images and suggests the usefulness and potential of eZIS.

Aging↗

PubMed1995

Grading of transitional cell bladder carcinoma by image analysis of histological sections.

Image analysis of histological sections was used to achieve a more objective malignancy grading of transitional cell carcinoma of the bladder. Images from Feulgen-stained sections from a clinical material of 197 tumours were analyzed. Features at various levels of analysis, e.g. mainly related to individual objects, neighbouring objects and the entire image, were analyzed. The features used were based on relational rather than individual nuclear features. With this technique, typical tissue architecture and degree of order/disorder can be described. These characteristics were compared by means of multivariate statistical methods with the subjective grading of the pathologists at our institution. This comparison provided the best subset of features and the agreement between the subjective and computer-based classification was 73%. The size, orientation and variation of the grey scale of the nuclei were particularly powerful. On a continuous scale, from grade 1 to 2A and 2B to 3, the four grades formed two distinct classes, low and high grade. In this distribution, the intermediate grade 2A was identified as a displaced grade 1 and the other intermediate grade 2B as a displaced grade 3.

Carcinoma, Transitional Cell↗

PubMed1992

[Image-analysis of salivary tumors].

The image analysis of 49 cases of salivary tumors showed that the cellular DNA content of most tumors (42/49) was diploidy and near diploidy. The heteroploidy rate was only 14.29% (7/49), which may be related to such clinical behaviors of salivary tumors as slow growth, relative low invasivity and metastatic ability. Among these morphologic parameters, nuclear perim and form PE were closely correlated with the malignancy of salivary tumors, the former was positive and the latter was negative. The insignificant nuclear heteromorphous appearance of adenoid cystic carcinoma and its relatively high cellular DNA content may be related to strong invasivity and metastatic ability. In conclusion, these parameters of DNA content and nuclear morphology supplied by image analysis are of value in the diagnosis of salivary tumors.

Adenocarcinoma↗

PubMedDate not supplied

An image analysis workstation designed for multiple users: application of quantitative digital imaging techniques to electron microscopy.

The purpose of the present study is to describe the setup of an image analysis workstation designed for multiple users, and to show the application of digital imaging techniques to the analysis of electron microscopic images. The image analysis system consists of a conventional light microscope mounted on a table-top, vibration-free platform, a light box for viewing negatives, two separate video cameras, a switch box, a video monitor, a digitizing tablet, a computer, and morphometric software packages. The system can quantitate the amount that each of the 256 gray levels contributes to the image, perform morphometric analysis (eg, shape and size) on individual gray level-defined subimages, and perform statistical analysis. Each operator has access to his or her own data and program setups through the use of 21.4-Mb removable Bernoulli cartridges. This setup for multiple users prevents the cluttering of the hard drive of the computer and avoids the possibility of accidentally removing the stored data of another user. The quantitative capabilities of the digital imaging system is demonstrated using an image of a normal lymphocyte and an apoptotic cell (ie, a cell which has undergone programmed cell death), both captured on the same electron microscopic negative. A comparison of the histograms of nuclear densities determined for these two cells reveals subtleties in gray level distribution not appreciated by the naked eye.

Clinical Laboratory Techniques↗

PubMed1999

Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis.

Simultaneous detection of two different antigens on paraffin-embedded and frozen tissues can be accomplished by double immunohistochemistry. However, many double chromogen systems suffer from signal overlap, precluding definite signal quantification. To separate and quantitatively analyze the different chromogens, we imported images into a Macintosh computer using a CCD camera attached to a diagnostic microscope and used Photoshop software for the recognition, selection, and separation of colors. We show here that Photoshop-based image analysis allows complete separation of chromogens not only on the basis of their RGB spectral characteristics, but also on the basis of information concerning saturation, hue, and luminosity intrinsic to the digitized images. We demonstrate that Photoshop-based image analysis provides superior results compared to color separation using bandpass filters. Quantification of the individual chromogens is then provided by Photoshop using the Histogram command, which supplies information on the luminosity (corresponding to gray levels of black-and-white images) and on the number of pixels as a measure of spatial distribution. (J Histochem Cytochem 47:119-125, 1999)

Actins↗

PubMed1999

[Immunohistochemical evaluation of the in vitro bromodeoxyuridine labeling index. 236 breast cancers studies by image analysis].

We studied with computerized image analysis 236 breast cancer samplings after in vitro bromodeoxyuridine incorporation and immunohistochemical revelation. Labeling index values were compared with the usual prognostic factors and with the other studies in the literature. We established a positive correlation between labeling index and tumor size, histoprognostic grading, phase S and DNA index. A high labeling index was correlated with the absence of hormonal receptors but not correlated with the other prognostic factors. These results on tumor kinetics are similar to those obtained by flow cytometry and from other studies in the literature. However, this technic using optical microscopy allows for reliable selection of tumoral cells. Furthermore, the semi-automated image analysis provides an objective and reproducible evaluation of the labeling index.

Adult↗

PubMed1989

Comparison of image analysis of imprints with flow cytometry for DNA analysis of solid tumors.

Quantitative analysis of cellular DNA content may be clinically useful for several solid tumors. The technology for this analysis by flow cytometry or image analysis has existed for several years but has not been widely used, except in a handful of specialized research institutions. Recently, however, relatively inexpensive image analyzers intended for use by hospital pathologists have been introduced that can analyze DNA content from cytology or imprint specimens which are readily obtainable from solid tumors. We report here an assessment of this technology for analysis of tumor imprint specimens, using flow cytometry of tissue blocks as the standard for comparison. We used image analysis equipment on Feulgan-stained imprint preparations from 31 tumors and compared the histograms with those obtained by flow cytometric analysis of archival tissue blocks from the same tumors. The ploidy descriptors (diploid, tetraploid, and aneuploid) were concordant for the two methods in 27 specimens, with three specimens yielding discordant results and one specimen considered unevaluable by image analysis. The image analysis method using imprints appeared to have several advantages over flow cytometry, including lower instrument cost, no need to dissociate paraffin blocks or fresh tissue, and ability to analyze very small samples. Somewhat lower resolution of the histograms, extremely localized tissue sampling, and possibly greater risk of occasionally obtaining unevaluable preparations were disadvantages. Microcomputer-based image analysis performed on imprints appeared to be a viable alternative to flow cytometric analysis of tissue blocks for quantitative DNA analysis of tumor specimens.

Aneuploidy↗

PubMed1993

Quantitative analysis of dystrophin gene amplification products using a PC-based image analysis system.

A PC-based image analysis system for gel photographs of DNA gel patterns was developed. It was originally designed as a general-purpose, low-cost, yet high-performance system for wide applications in the biomedical area. In this study, we performed analysis of gel images obtained by polymerase chain reaction (PCR) amplification. The system employs a high-resolution CCD camera that can accurately measure grayness of the gel photographs for quantitative analysis of PCR products. The target DNA (exon 52 of the dystrophin gene), which had been found to be deleted in some patients with Duchenne/Becker muscular dystrophy (DMD/BMD), was amplified for the female family members together with the control DNA (exon 60) as a reference. The ratio of the target DNA to the control DNA was determined from PCR products to identify the carrier status of this disease by means of gene dosage. We conclude that 'the PC-based image analysis system' was useful for quantitative deletion analysis of DMD/BMD heterozygotes.

Adult↗

PubMed1989

Computer image analysis of toxic fatty degeneration in rat liver.

Fatty degeneration of the liver is one of the most frequently observed pathological changes in the experimental estimation of the toxicity of chemical compounds. The intensity of this kind of damage is most often detected by means of a generally accepted scale of points, whereas the classification is performed according to the subjective "feeling" of the pathologist. In modern pathological diagnostics, computer analysis of images is used to perform an objective estimation of the degree of damage to various organs. In order to check the usefulness of this kind of method, comparative biochemical and morphometrical studies were undertaken in trichloroethylene (TRI)-induced fatty degeneration of the liver. TRI was administered to rats intragastrically, in single doses: 1/2; 1/3; 1/4; 1/6 and 1/18 DL50. 24 hours after the administration, the animals were sacrificed. The content of triglycerides in the liver was determined according to Folch et al. (1956). Simple lipids in the histochemical samples were detected by means of staining with a lipotropic, Fat Red 7B. The area of fatty degeneration was estimated in the microscopic samples by the use of an automatic image analyser IBAS 2000 (Kontron). The morphometrical data concerning the area of fatty degeneration in the liver amplified a high degree of correlation with the content of triglycerides (r = 0.89) and the dose of TRI (r = 0.96). The degree of correlation between the biochemical data and the dose of TRI was 0.88. The morphometrical studies performed have proved to be of great use in estimating the degree of fatty degeneration in the liver. This method enables precise, quantitative measuring of this sort of liver damage in the material prepared for routine histopathological analysis. It requires, however, the application of a specialized device for quantitative image analysis.

Animals↗

PubMed2003

A longitudinal study of brain morphometrics using quantitative magnetic resonance imaging and difference image analysis.

Serial quantitative magnetic resonance imaging (MRI) allows the detection of subtle volumetric changes in brain volume. We used serial volumetry and voxel-based difference image analysis to quantify and characterize longitudinal changes in the hippocampus, cerebellum, and neocortex in younger and middle-age individuals. Paired volumetric MRI brain scans 3.5 years apart were performed on 90 healthy subjects 14 to 77 years old. Quantitative assessment of registered images included hippocampal volumetry, cerebellar volumetry, and automatically determined regional brain volumes. Longitudinal volume changes in three age epochs (<35, 35-54, >54 years) were compared and neocortical changes beyond regions of interest were visualized using filtered difference images. Cross-sectional analysis revealed a significant association between age and reduction in all brain volumes except hippocampal volume. Changes in normalized hippocampal and white matter volume were significantly different among the three groups. Individual analysis revealed 5 subjects with significant longitudinal volume changes lying outside the normative range. Difference image analysis showed global involutional changes in the >54 age group. Our findings suggest that cross-sectional observations in intracranial volume, cerebellar volume, and gray matter volume are likely to reflect uniform rates of volume loss or secular changes. Accelerated brain atrophy was seen from the age of 35-54 and increased rates of hippocampal atrophy from the age of 54. Our findings emphasize the importance of controlling for age effects when studying pathological brain changes over a wide age range.

Adolescent↗

PubMed2003

Clinical monitoring of ocular physiology using digital image analysis.

AIM: To examine the use of image analysis to quantify changes in ocular physiology. METHOD: A purpose designed computer program was written to objectively quantify bulbar hyperaemia, tarsal redness, corneal staining and tarsal staining. Thresholding, colour extraction and edge detection paradigms were investigated. The repeatability (stability) of each technique to changes in image luminance was assessed. A clinical pictorial grading scale was analysed to examine the repeatability and validity of the chosen image analysis technique. RESULTS: Edge detection using a 3 x 3 kernel was found to be the most stable to changes in image luminance (2.6% over a +60 to -90% luminance range) and correlated well with the CCLRU scale images of bulbar hyperaemia (r=0.96), corneal staining (r=0.85) and the staining of palpebral roughness (r=0.96). Extraction of the red colour plane demonstrated the best correlation-sensitivity combination for palpebral hyperaemia (r=0.96). Repeatability variability was <0.5%. CONCLUSIONS: Digital imaging, in conjunction with computerised image analysis, allows objective, clinically valid and repeatable quantification of ocular features. It offers the possibility of improved diagnosis and monitoring of changes in ocular physiology in clinical practice.

Journal Article↗

PubMed1998

Stain removal efficacy: an in vitro evaluation using quantitative image analysis.

OBJECTIVE: This study developed a computer image analysis technique as a quantitative means to measure changes in dental stain after brushing with various dentifrices. METHOD AND MATERIALS: Enamel specimens with naturally occurring mature stain were cut from bovine incisors. The specimens were subjected to in vitro toothbrushing with one of the four tested groups, consisting of two dentifrices that make claims of stain removal (Aquafresh Whitening and Rembrandt Sensitive), a regular dentifrice, (Aquafresh Triple Protection), and water. Digital images of stain specimens were recorded under standardized lighting conditions and analyzed with an image analysis software. The area-intensity stain determinant, which accounted for the reflected intensity and the corresponding areas of stain, was computed. Stain removal efficacy was calculated based on the difference in area-intensity stain determinant before and after brushing. RESULTS: Brushing with any of the tested dentifrices removed more stain than did brushing with water alone. The finding that brushing with a regular dentifrice resulted in higher stain removal efficacy than brushing with water seems to indicate a role for abrasivity. Aquafresh Whitening had a higher stain removal efficacy than did Rembrandt for the removal of mature calcified stain used in this study. However, there were certain stains that none of the dentifrices removed. CONCLUSION: Computer image analysis provides an objective and quantitative measurement to distinguish in vitro stain removal efficacy of dentifrices.

Analysis of Variance↗

PubMed1990

[Method of automatic recording and analysis of leukocyte migration based on the image analysis system].

The method of automatic registration and of the analysis of neutrophils' mobility with the use of the Image Analysis System is described in this paper. The authors developed an algorithm of cells identification which makes it possible to carry out cadre-by-cadre observing the shift of each cell's centre of gravity with the time interval between cadres more than 15 seconds. This algorithm also allows to measure areas, perimeters and orientation of cells and their changes. Looking after the shifts of the cells' centres of gravity allows to describe the cells' paths in the form of the following dependences: path-by-time, shift-by-time, angle of vector of shift alteration. Observing a great number of cells at the same time allows to get true statistical picture of neutrophils' activity and distribution of cells' activity in population of neutrophils during only some minutes of registration. It is shown that each cell in the population has its own constant velocity.

Algorithms↗

PubMed2003

[An improved imaging analysis for quantitative measurement of brain slice volume].

OBJECTIVE: To improve computer-assisted imaging analysis for quantitatively measuring brain slice volume of rats and mice in comparison with conventional measuring methods,and to evaluate its usefulness in assessment of focal cerebral ischemia. METHODS: The accurate volumes of rat and mouse brain slices were measured by weight and special gravity measuring. The areas of brain slices were measured by imaging analysis, then the slice volumes of right and left hemispheres were calculated by multiplying the adjusted thickness of the slices. In addition, the brain slice volumes of right and left hemispheres from focal cerebral ischemic mice were compared to assess ischemic injury using the imaging analysis. RESULT: Area measurement by computer-assisted imaging analysis was linear with different accurate areas (r=1.000). Slice volumes measured by imaging analysis correlated well with the accurate volumes measured by special gravity method, r=0.809 (n=45, P<0.001) in rats, and r=0.844 (n=74, P<0.001) in mice. The brain volumes in ischemic hemispheres were larger than in non-ischemic hemispheres in ischemic mice. CONCLUSION: Computer-assisted imaging analysis can measure the brain slice volumes accurately and compare right and left hemisphere volumes quantitatively.

Animals↗