Microvessel density counting in breast cancer. Slides vs. digital images

OBJECTIVE: To develop a program to assist the pathologist in the acquisition and evaluation of digital images to determine microvessel density (MVD) in tissues. STUDY DESIGN: Ten cases of breast cancer with a high degree of neovascularization were selected. A standard immunohistochemical method was used to highlight the microvessels (monoclonal anti-factor VIII, avidinbiotin-peroxidase complex method). Two pathologists (one senior [S] and one junior [J]) evaluated four areas of highest neovascularization ("hot spots") in the tumors. Microscopically MVD was determined in four chosen areas (400:1). From the center of each area two digital images were acquired at a magnification of 200:1. All counts made by microscopic observation were compared with those made on the digital images. To compare MVD counting at different resolution, two sets of images at different sampling densities (320 x 240 and 1,600 x 1,200) were assessed by the two pathologists. RESULTS: We obtained a good correlation (r = .98 for S and .96 for J) between the MVD counts obtained at the microscope (192.8 MV/mm2 [mean of S] and 181.8 MV/mm2 [mean of J]) and the MVD counts from digital images (153.2 MV/mm2 [mean of S] and 171.0 MV/mm2 [mean of J]) at high resolution. The counts were lower for digital images at lower sampling density (125.0 MV/mm2 [mean of S] and 78.2 MV/mm2 [mean of J]). With low-resolution digital images only S maintained a good correlation (r = .96 for S and .34 for J) with the microscopic evaluation of MVD. Interobserver analysis showed a good correlation (r = .82 for the microscope and r = .78 for the digital images) of MVD evaluated either at the microscope or in high-resolution digital images. CONCLUSION: We demonstrated the functionality and usefulness of our program in performing MVD evaluation. Considering the capabilities of the program to store all images and microvessel marks and the reliability of MVD evaluation based on digital images, we consider this program the first step toward fully automated MVD assessment.     

Quantification of the magnification and distortion effects of a pediatric flexible video-bronchoscope

Background

Flexible video bronchoscopes, in particular the Olympus BF Type 3C160, are commonly used in pediatric respiratory medicine. There is no data on the magnification and distortion effects of these bronchoscopes yet important clinical decisions are made from the images. The aim of this study was to systematically describe the magnification and distortion of flexible bronchoscope images taken at various distances from the object.

Methods

Using images of known objects and processing these by digital video and computer programs both magnification and distortion scales were derived.

Results

Magnification changes as a linear function between 100 mm (×1) and 10 mm (×9.55) and then as an exponential function between 10 mm and 3 mm (×40) from the object. Magnification depends on the axis of orientation of the object to the optic axis or geometrical axis of the bronchoscope. Magnification also varies across the field of view with the central magnification being 39% greater than at the periphery of the field of view at 15 mm from the object. However, in the paediatric situation the diameter of the orifices is usually less than 10 mm and thus this limits the exposure to these peripheral limits of magnification reduction. Intraclass correlations for measurements and repeatability studies between instruments are very high, r = 0.96. Distortion occurs as both barrel and geometric types but both types are heterogeneous across the field of view. Distortion of geometric type ranges up to 30% at 3 mm from the object but may be as low as 5% depending on the position of the object in relation to the optic axis.

Conclusion

We conclude that the optimal working distance range is between 40 and 10 mm from the object. However the clinician should be cognisant of both variations in magnification and distortion in clinical judgements.     

Microscope densitometer system for point measurement of autoradiograms.

A photometric system for measuring optical densities generated by tissue autoradiograms has been adapted to an ordinary light microscope. The optics of the microscope are used to direct the light transmitted through the autoradiogram to ocular-mounted photoconductive cells coupled to a bridge amplifier. Readout is on a digital panel meter. The integrated area analyzed varies between 33.16 to 0.02 0.02 mm2 depending on the objective magnification. The system is linear over a range of optical densities from 0.5 to 0.05.     

无损光学法测量人胃粘膜/粘膜下层组织的光衰减特性

研究了人正常胃粘膜及粘膜下层组织对640 nm,690 nm,740 nm,790 nm,840 nm和890 nm波长的钛宝石激光的光衰减特性以及光学穿透深度,实验采用激光斜入射式空间分辨反射光和CCD探测器以及非线性拟合确定组织光学特性。结果表明:人正常胃粘膜及粘膜下层组织对六个波长的激光的有效衰减系数和光学穿透深度都是随着激光波长的变化而变化的。其有效衰减系数的最大值在640 nm,其值为1.12 mm-1,最小值在790 nm,其值为0.901 mm-1,最大差异在790 nm和890 nm之间,其值为19.9%,最小差异在690 nm和740nm之间,其值为2.83%。其光学穿透深度的最大值在790 nm,其值为1.11 mm,最小值在640 nm,其值为0.890 mm,最大差异在640 nm和790 nm之间,其值为24.7%,最小差异在690 nm和740 nm之间,其值为2.97%。     

Measurement of capillary basement membrane thickness by magnification on a rear opaque projection screen

2190 measurements of capillary basement membrane thickness were obtained from the images of magnified micrographs using a rear opaque projection screen. This method proved to be reproducible and reliable. The electron microscope magnification used, 2500x, permits sharp definition of structures in the resulting micrographs. Projection on the opaque rear screen magnified the image 16x, yielding a final magnification of 40,000x. This high final magnification of micrographs of good quality minimized the error in measurement. The use of a special dial caliper with pins attached allows an open field and decreases the subjective components of measurements. Moreover, this technique results in less stress for the persons performing the measurements.     

Confocal assay for invasion: use of propidium iodide fluorescence and laser reflectance to quantify the rate of migration of cells through a matrix

BACKGROUND: Most assays used to measure invasion are based on manual counting of the number of cells that have migrated completely through commercial coated filters. We describe here a confocal fluorescence-imaging method that can assess the relative rates of invasion of cells into a matrix. METHODS: After being seeded on the matrix and a period of incubation, the cells are fixed and treated with RNase. Propidium iodide is then added to stain the double-stranded DNA. A confocal microscope system is used to obtain high-resolution images of the red propidium iodide fluorescence and laser reflectance from optical sections at increasing depths in the matrix. The section with high laser reflectance marks the top of the matrix. RESULTS: Data were calculated as the total area of red fluorescence above background in each section and were plotted as a percentage of the summed fluorescent areas in all sections. CONCLUSIONS: Because the distance into the matrix of the nuclei can be calculated by measuring from the reflective upper surface of the matrix, the method is useful for assessing the rate of cell migration and for comparing the ability of different cells to invade through different matrices under varying conditions.     

Quantification of fluorescence in situ hybridization signals by image cytometry.

In this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluated. Quantification of the fluorescence ISH signals was performed using an epi-fluorescence microscope with a multi-wavelength illuminator, equipped with a cooled charge couple device (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimen. Specific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non-uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min-max filter) to estimate local background intensities. The choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of field. The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double-labeled probes.     

Vocal fold composition and early glottic carcinoma infiltration

ABSTRACT: BACKGROUND: Current imaging techniques provide only limited information pertaining to the extent of infiltration of laryngeal carcinomas into vocal fold tissue layers. Therefore, it is needed to seek the contribute to the body of knowledge surrounding examination and characterization in laryngeal carcinoma infiltration. METHODS: Excised larynges were collected from 30 male laryngectomy patients with an average age of 43.5 years (ranging 36 to 55 years) and history of smoking ([GREATER-THAN OR EQUAL TO]10 years) exhibiting T1, T2, or subglottal (normal vocal fold) carcinomas. Vocal folds were preserved via freezing or immersion in paraffin. The depth of the mucosa, submucosa, and muscular layers in both normal vocal folds and tumor tissues of afflicted vocal folds was measured. RESULTS: The average depths of the mucosa, submucosa, and muscular layers in normal vocal folds were 0.15 [PLUS-MINUS SIGN] 0.06 mm, 2.30 [PLUS-MINUS SIGN] 0.59 mm, and 2.87 [PLUS-MINUS SIGN] 0.88 mm, respectively. Infiltration measurements of T1 tumors showed a depth of 1.62 [PLUS-MINUS SIGN] 0.51 mm and 1.32 [PLUS-MINUS SIGN] 0.49 mm in frozen sections and paraffin-embedded samples, respectively. Similarly, T2 tumors showed a depth of 2.87 [PLUS-MINUS SIGN] 0.68 mm and 2.58 [PLUS-MINUS SIGN] 0.67 mm in frozen sections and paraffin-embedded samples, respectively. T1 and T2 tumors occupied 24.8 [PLUS-MINUS SIGN] 10 and 48.5 [PLUS-MINUS SIGN] 15 percent of the normal vocal fold depth, respectively. CONCLUSION: This data provides a baseline for estimating infiltration of laryngeal carcinomas in vocal fold tissue layers, of particular interest to surgeons. This information may be used to assess typical depths of infiltration, thus allowing for more appropriate selection of surgical procedures based on individual patient assessment.     

In vivo assessment of muscle fascicle length by extended field-of-view ultrasonography

The present study examined the reliability and validity of in vivo vastus lateralis (VL) fascicle length (L(f)) assessment by extended field-of-view ultrasonography (EFOV US). Intraexperimenter and intersession reliability of EFOV US were tested. Further, L(f) measured from EFOV US images were compared to L(f) measured from static US images (6-cm FOV) where out-of-field fascicle portions were trigonometrically estimated (linear extrapolation). Finally, spatial accuracy of the EFOV technique was assessed by comparing L(f) measured on swine VL by EFOV US to actual measurements from digital photographs. The difference between repeated VL L(f) measurements by the same experimenter was 2.1 ± 1.7% with an intraclass correlation (ICC) of 0.99 [95% confidence interval (CI) = 0.95-1.00]. In terms of intersession reliability, no difference (P = 0.48) was observed between L(f) measured on two different occasions, with ICC = 0.95 (CI = 0.80-0.99). The average absolute difference between L(f) measured by EFOV US and using linear extrapolation was 12.6 ± 8.1% [ICC = 0.76 (CI = -0.20-0.94)]; EFOV L(f) was always longer than extrapolated L(f). The relative error of measurement between L(f) measured by EFOV US and by dissective assessment (digital photographs) in isolated swine VL was 0.84% ± 2.6% with an ICC of 0.99 (CI = 0.94-1.00). These results show that EFOV US is a reliable and valid method for the measurement of long muscle fascicle in vivo. Thus EFOV US analysis was proven more accurate for the assessment of skeletal muscle fascicle length than conventional extrapolation methods.     

用于海洋原位浮游生物探测的同轴数字全息显微技术研究

本文将数字全息与显微成像技术相结合,设计搭建了一套数字全息显微系统,用于对浮游生物进行光场获取。基于该系统获取的光场信息,通过在不同景深对图像进行再现,既可以获到单一浮游生物的清晰图像,也可以获得一定水体内浮游生物微粒在海水中的三维分布情况。通过实验测得系统分辨率可以达到7．8微米,景深可以达到10毫米,优于一般光学显微镜的技术指标。研究结果表明优势明显的数字全息显微系统是一种适合海洋原位探测的浮游生物研究的有效方法。基于数字同轴显微系统的水下仪器开发将是下一步工作的努力方向。     

Conidium production by insect pathogenic fungi on commercially available agars

AIMS: Conidium production by three species of insect pathogenic fungi, Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii, was assessed on various depths and types of commercially available agars. METHODS: Conidium production was assessed after 14 d of growth on commercially available media as well as at three different agar depths. RESULTS: Metarhizium anisopliae and B. bassiana isolates showed greatest conidium production on potato dextrose agar (PDA) at a depth of 2 mm, whereas V. lecanii showed greatest conidium production on yeast extract-peptone-dextrose agar (YPDA) regardless of agar depth. Optimum conidium production for M. anisopliae and B. bassiana was not only dependent upon the isolate used but also on the medium type and agar depth. SIGNIFICANCE AND IMPACT OF THE STUDY: Conidia are the infective structures for insect pathogenic fungi and this study suggests a rationale basis for consistent conidium production for laboratory and commercial practices.     

Intercondylar distances of the human temporomandibular joints

In a sample which included subjects of the Croatian population we made measurements of intercondylar distances between the temporomandibular joints in radiographs. A total of 101 subjects of both sexes ranging in age from 20 to 80 years, mostly residents in Zagreb, were evaluated. We measured the intercondylar distances from the condyle centers in the postero-anterior cranial radiographs which had previously been examined and traced on acetate paper. The measuring points were digitized prior to measurements. A special system of coordinates was devised for each radiograph. The results of our measurements were assessed by ANOVA analysis. The intercondylar distance between the two temporomandibular joints was within the range of 110 and 145 mm, with the mean of 126 mm. In men the intercondylar distance was within the range of 116 and 145 mm, with the mean of 130.2 mm. In women the distance ranged from 110 to 138 mm, with the mean of 123.5 mm. There was a significant difference between the two sexes. From a review of the literature, it is apparent that the results of our measurements do not support the results of similar studies assessed by a number of researchers in other countries. The intercondylar distance in the Croatian sample was 5.25% larger than the maximal values of the same parameters in other populations suggesting larger craniofacial skeletons. The development of this radiographic assessment method should improve evaluation of subjects seeking treatment.     

Discrimination of the redfish (Sebastes mentella) stock components in the Irminger Sea and adjacent waters based on meristics,morphometry and biological characteristics

Redfish Sebastes mentella samples were collected in 2004 and 2005 during commercial cruises to the Irminger Sea on board the Polish vessel M/T ‘Wiesbaden’. Ichthyological studies included length and weight measurements, sex, gonad maturity stages and age determinations. Meristic and morphometric measurements were performed on digital images of the redfish. Comparison of the pelagic redfish from the northeastern (depths deeper than 500 m) and southwestern (depths shallower than 500 m) fishing grounds of the Irminger Sea indicates a number of differences including spatial and vertical distribution, ambient temperature, length and age composition. Moreover, 12 morphometric and 4 meristic characters differed significantly between fish samples from these two areas. Results of Cluster Analysis showed the clear grouping of samples into that of the ‘oceanic’ component (depth 300–450 m) and the ‘deep sea’ component (depth 550–800 m). These results were confirmed by Principal Component Analysis, which revealed the separation of samples into two catch depth groups. The share of fish allocated by Discriminant Analysis into the pelagic ‘deep sea’ component in the northeastern area was nearly 92%, while the ‘oceanic’ component was dominant in the southwestern area and comprised more than 88% of the fish. Cluster and Principal Component analyses suggest that the ‘oceanic’ component is a more homogeneous group than the ‘deep sea’ component. These results support the management units recently established by ICES.     

Microscope Densitometer System for Point Measurement of Autoradiograms

A photometric system for measuring optical densities generated by tissue antoradiograms has been adapted to an ordinary light microscope. The optics of the microscope are used to direct the light transmitted through the autoradiogram to ocular-mounted photoconductive cells coupled to a bridge amplifier. Readout is on a digital panel meter. The integrated area analyzed varies between 33.16 to 0.02 mm² depending on the objective magnification. The system is hear over a range of optical densities from 0.5 to 0.05.     

High-resolution cytometry of FISH dots in interphase cell nuclei.

BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSCM) provide indispensable tools for measuring large number of cells with low resolution. Confocal microscopy, on the other hand, is used for measuring small number of cells with high resolution. In this paper, we present a reasonable compromise between the two extremes. METHODS: We have developed a completely automated, high-resolution system (high-resolution cytometer, HRCM) capable of analyzing microscope slides with FISH-stained interphase nuclei in two dimensions as well as in three dimensions using a fully motorized epi-fluorescence microscope and a cooled digital CCD camera fully controlled by a high-performance computer which performs both acquisition and related on-line image analysis. The images of different dyes are acquired sequentially using highly specific filters and superimposed in computer memory. For each nucleus and each hybridization dot, user-selected attributes (such as position, size, intensity, etc.) are computed off-line using another processor or computer connected with a network. RESULTS: Using HRCM, it is possible to analyze multi-color preparations including UV-excited dyes as well as repeatedly hybridized preparations reacquiring individual nuclei. The speed of the acquisition and analysis is about 50 nuclei per minute in two dimensions and 1 nucleus per minute in three dimensions, but depends on the density of nuclei on the slide; the precision of the lateral and axial measurements is approximately 100 nm. CONCLUSIONS: Thus, using overnight acquisition, quantities comparable to those of FCM or LSCM measurements can be analyzed with an accuracy comparable to confocal microscopy. HRCM is suitable for a number of clinical and scientific tasks: routine diagnostics, follow-up of therapy, studies of chromatin structure, and many other different aspects of cell research.     

Morphological characteristics of the acetabulum

The aim of this research was to accurately measure the surface of the semi lunar articular surface of the pelvis (facies lunata acetabuli) and the variability of the acetabular geometry, as well as to determine the correlation between measured parameters. 30 macerated anatomical specimens of pelvic bones were measured. The radius and depth were measured in the classical way, while cartilaginous surface area was measured using small fragments of measuring paper to avoid errors in measurement due to the curvature of the surface. Computerized calculations provided accurate surface values. In our research, facies lunata acetabuli measured 2294+/-329 mm2. Diameter of the opening of the acetabulum measured 25.8+/-1.9 mm. Acetabular depth was 30+/-3.2 mm. Correlations between the surface area of the facies lunata acetabuli and the radius of the acetabular opening curvature (r=0.71), surface area of the facies lunata acetabuli and the depth of the acetabulum (r=0.80) and the radius of the acetabular opening curvature and the depth of the acetabulum (r=0.80) were confirmed. For precise assessment of the facies lunata acetabuli surface area, the simplest and the cheapest method is the method of measurement using small fragments of measuring paper and software analysis. There is a significant correlation between the depth, opening of the acetabulum and surface area of the facies lunata.     

Telediagnosis of transbronchial fine needle aspirations--a feasibility study.

AIM: To analyze the diagnostic accuracy of telediagnostic procedures for transbronchial fine needle aspirations. MATERIAL AND METHODS: A double blind study was performed on cytological slides of 54 randomly selected cases with transbronchial fine needle aspirations. The slides were digitized using a Leica digital camera DC100 mounted on a microscope Leica Laborlux S, and analyzed by an experienced pathologist without knowing the definite diagnosis or any additional clinical data. The diagnoses stated by analyzing the digital images were compared to the final conventional diagnoses. In addition, the duration of the digital diagnosis, used magnifications, and difficulties for correct sampling were documented. RESULTS: The "digital" diagnoses of the 54 cases were all in general agreement with the definite diagnoses. No wrong positive or wrong negative case in respect to malignant/non-malignant (31/23) or to small cell/non-small cell (9/22) occurred. The performance of a digital diagnosis lasted for 115 s at average (15-260 s), and is significantly longer compared to that of conventional fine needle aspiration judgement (20 s). The screening magnification was commonly set to x2.5, that for definite diagnostic analysis x40. Benign diseases (2 tuberculosis and 3 sarcoidosis cases) were correctly classified. CONCLUSIONS: Telepathology systems can probably be used for fine needle aspiration analysis without major diagnostic errors. Their use can improve the endoscopic sampling and avoid second anesthesia when missing the lesion of request during the first examination.     

Description of the Photoperiodic Control of Larval Burrowing in the Blowfly Lucilia Cuprina: a Novel Index for Photoperiodic Research

The photoperiodic control of larval burrowing depth in the Australian sheep blowfly Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) was investigated by measuring burrowing depth under controlled laboratory photo-periods. The results demonstrated that larvae exposed to long photoperiods (LD 18:6) burrowed to deeper depths than those in shorter photoperiods (LD 12:12), and that this behavior was induced during the third instar stage. The ecological significance of this behavior is discussed, as are the ways in which daylength is measured and depth assessed. The use of burrowing depth could prove to be a novel index of a photoperiodic response and provide a far simpler approach to the study of photoperiodism in certain insect species. (Chronobiology International, 14(3), 247–252, 1997)