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Automated Detection and Delineation of Mitochondria in Electron Micrographs of Human Skeletal Muscles
Abstract
Morphometric measurements of mitochondria in human skeletal muscles provide useful information relating to tissue oxidative energy production, nutrition, exercise, and aging. Morphometric data such as area, perimeter, long axis, and short axis can be obtained by delineating individual mitochondria in electron micrographs. However, manual counting and delineating of individual mitochondria is a formidable task. The purpose of this study was to develop a fully automated computer algorithm for quantifying mitochondrial morphometry in electron micrographs. The algorithm locates mitochondria with a two-dimensional matched filter and then traces the borders of individual mitochondria. The delineation is accomplished by edge detection along radial lines launched outwards from the center of each mitochondrion. Shape descriptors applied to delineated mitochondria are used to reject likely false-positive selections. The results show that the fully automated algorithm detects mitochondria with a false-positive rate of 2% and a false-negative rate of 36%. The errors are easily and rapidly corrected by user intervention using a second semiautomated delineation algorithm. Morphometric measurements collected with the automated algorithm are equivalent to those obtained manually by human experts. The algorithm significantly improves the speed of image analysis and it also provides copious quantities of high-quality mitochondrial morphometric data.
... According to studies of muscle cell mitochondria, electron microscopy images of longitudinal sections are often used. Guo et al. [4] proposed an automated algorithm * e-mail: zdenka.uhrikova@gmail.com for mitochondria detection, based on application of a twodimensional matched filter to trace the borders of mitochondria. ...
... Vendelin et al. [11] found that mitochondria are arranged in crystal-like patterns and they also evaluated distances between pairs of mitochondria. In fact, both [4] and [11] analyze only spot-shaped mitochondria, and neglected long ones. ...
In this paper we describe a new approach to quantita-tive measurements on muscle cell images. Our algo-rithm processes gray scaled images obtained from electron microscopy (12000× magnification). By means of dis-crete Fourier transform we detect the orientation of mus-cle fibers. Afterwards, applying our thresholding method, we detect boundaries between constituents of myofibrils (A band, I band and Z line). From these boundaries we estimate lengths of each myofibril constituent, since these lengths define the type of a muscle cell. Finally, we com-pare data measured by our algorithm, with data estimated manually.
... Each line consists of a discrete number of intensities sampled at a certain step size along the radial line. [4] This sampling does not actually begin until a specified "dead zone" has been cleared, which is a predefined circular region in which no samples are taken in order to avoid mistaking blood vessels for disc and cup edges (the vessels converge right near the center of the optic disc). The sampling locations are described by the following formulas: (1) and (2), and refer to the and pixel coordinates of the center point, ( ) and ( ) refer to the and coordinates of sample j along a radial line projected at angle to the horizontal. ...
The aims of this project were twofold: (1) to develop an accurate, yet simple and efficient algorithm that estimates the optic cup-to-disc ratio from input retinal images (a critical feature in the evaluation of glaucoma), and (2) to implement this algorithm in a graphical user interface (GUI) that can aid optometrists and other trained professionals in this characterization process. The first aim addresses the possibility of having this diagnostic tool implemented in devices that can be transported to areas currently devoid of appropriate medical care at low prices. Both goals have been completed successfully. The developed algorithm works as follows: by selecting the center point of the optic disc and optic cup on a retinal image, radial lines are projected outward in a circular pattern that detect the edges by determining the greatest change in pixel intensity. The points of greatest difference are connected to form polygonal approximations of the desired features. The areas of the polygons are calculated and the ratio between the two areas is determined, providing a reliable estimate to the relevant cup-to-disc ratio.
... The main portion of the algorithm deals with edge detection and delineation of the pulmonary artery crosssectional borders. The algorithm implemented is largely based on a previously developed algorithm for delineating mitochondria in electron micrographs in human skeletal muscles [5]. Initially, a seed point within the cross-sectional area of the artery is needed: when in the 2D viewer mode of OsiriX, the user can select a point (the seed point) using the pull down mouse option, as shown in Fig. 1. ...
The purpose of this project was to develop a software tool for studying the dynamic changes of the cross-sections of the pulmonary arteries from computer-tomography (CT) scans. The software for analyzing CT images in the DICOM format was developed using Cocoa, the Apple's object-oriented C++ application programming interface (API) for the Mac OS X operating system, specifically using Mac OS X Lion. The software was developed as a plugin for OsiriX, which is an open-source, widely used DICOM viewer. The main algorithm for detecting the artery border first establishes a seed point within the cross-sectional area of the pulmonary artery, based off of an initial user click. Then the artery border is detected in an automated way by launching 24 radial lines from this seed point. In a similar fashion the border detection continues onto the subsequent frames.
An optical flow gradient algorithm was applied to spontaneously forming networks of neurons and glia in culture imaged by fluorescence optical microscopy in order to map functional calcium signaling with single pixel resolution. Optical flow estimates the direction and speed of motion of objects in an image between subsequent frames in a recorded digital sequence of images (i.e., a movie). Computed vector field outputs by the algorithm were able to track the spatiotemporal dynamics of calcium signaling patterns. We begin by briefly reviewing the mathematics of the optical flow algorithm, and then describe how to solve for the displacement vectors and how to measure their reliability. We then compare computed flow vectors with manually estimated vectors for the progression of a calcium signal recorded from representative astrocyte cultures. Finally, we applied the algorithm to preparations of primary astrocytes and hippocampal neurons and to the rMC-1 Muller glial cell line in order to illustrate the capability of the algorithm for capturing different types of spatiotemporal calcium activity. We discuss the imaging requirements, parameter selection and threshold selection for reliable measurements, and offer perspectives on uses of the vector data.
Morphometrie ist die quantitative Erfassung und vergleichende Analyse biologischer Formen zur Auffindung struktureller Unterschiede zwischen Untersuchungsobjekten. Dabei ist bei praktischer Anwendung etablierter Methoden der personelle und materielle Aufwand durch die manuelle Identifizierung und Umfahrung der Strukturgrenzen sowie die nachfolgende Digitalisierung per Zeichentablett häufig ein begrenzender Faktor für umfangreichere Untersuchungen an großen Populationen. Zielsetzung dieser Arbeit war die Entwicklung eines Computer-Programms, das diese Probleme überwindet und in einer vergleichenden Testung am Beispiel des Nucleus rotundus bei Hühnervögeln seine Reliabilität beweist. Dazu wurde bei 30 Hühnervögeln (3 Rassen à 10 Tiere, Geschlechterverhältnis 1:1) der Nucleus rotundus über ein Lichtmikroskop mit Zwischentubus für Bildeinspiegelung umfahren. Hierzu wurde der Logitech IO-Pen verwendet. Dieser ist ein Hybrid-Stift, bestehend aus einer klassischen, dokumentenechten Tintenmine und einer digitalen Optik, der die unverfälschte, zeitgleiche digitale Speicherung mit ihm erstellter handschriftlicher Dokumente und ihre anschließende Übertragung auf den PC ermöglicht. In Kombination mit einem Spezialpapier wird die Position seiner Mine immer eindeutig und unabhängig vom Winkel und der Ausrichtung der Optik registriert. Die neu entwickelte Software NucleoScope steuert den Prozess der Datenanalyse der vom IO-Pen generierten Bilder, rekonstruiert aus den Ergebnissen der Einzelflächen die zugehörigen Volumina und überträgt diese in ihre interne Datenbank. Alle Datensätze können zur statistischen Weiterverarbeitung in allen gängigen Dateiformaten exportiert werden. Abschließend wurde ein quantitativer Vergleich der Ergebnisse der neuen, automatisierten und digitalen mit der etablierten Vorgehensweise durch die statistische Methode nach Bland und Altman (1986) durchgeführt. Dabei zeigten sich über die Gesamtheit aller umfahrenen Einzelschichten keine signifikanten Unterschiede zwischen beiden Methoden. Es lagen 95% der mit IO-Pen und NucleoScope ermittelten Volumina unterhalb einer Abweichung von 1% bezogen auf den Referenzwert. Der methodisch notwendige Zeitaufwand wurde insgesamt um mehr als 60% reduziert. Es konnte gezeigt werden, dass der neue Algorithmus mittels IO-Pen und NucleoScope bezogen auf die Referenzmethode gleichwertige Ergebnisse liefert und diese zu ersetzen vermag. Der bedeutendste Vorteil jedoch ist die mögliche Vollautomatisierung des Analyseprozesses, so dass die hier vorgestellte Alternative gegenwärtig die schnellste und preiswerteste Methode zur Erfassung von Volumina beliebiger anatomischer Strukturen ist. Objectives: To develop a fast and accurate digital technique for the extraction of quantitative volumetric data from histological sections and to validate the results obtained by this new method. Methods: An entirely new algorithm was proposed based on a Digital Pen and Paper (DP&P) setup in conjunction with a newly programmed software called NucleoScope. The software allowed for a totally automated and user-independent work-flow once the identification and delineation process of the structure of interest was finished by the examiner. Different morphological operators for image analysis were tested, optimized and incorporated into the software. In a second step, a statistical analysis based on the method proposed by Bland and Altman for assessing agreement between two methods was carried out. The object of interest for the comparison was the Nucleus rotundus of 30 animals (Gallus gallus). For this comparison, the delineations were also digitized on a digitizer-tablet, which is still considered the reference method. Results: Measurements taken with the new algorithm may be 3.667 % above or 3.533 % below the digitizer tablet. There was no relevant systematic difference between the measurements. Conclusions: The agreement between methods was absolutely acceptable for morphometrical purposes. Optimizations in NucleoScope and the utilization of a DP&P setup for delineating structures of interest generated a user-independent work-flow which allowed for a reduction of the methodical time-effort by more than 50%.
A morphometric analysis was performed on horse muscle tissue to quantify mitochondrial distribution relative to capillaries. Samples of M. vastus medialis, M. semitendinosus, M. masseter and M. cutaneus thoracicus were preserved in a glutaraldehyde fixative for electron microscopy, or frozen for biochemical and histochemical analysis. These four muscles varied from highly oxidative in type, consisting nearly completely of type I fibres, in masseter, to highly glycolytic, primarily type IIb fibres, in cutaneus. In all four muscles, mitochondria were found in highest volume density near capillaries at the fibre border, with a sharp decline in volume density towards the fibre centre. This distribution was independent of myoglobin concentration, muscle fibre type and the activities of three key metabolic enzymes, citrate synthase, 3-OH-acyl-CoA dehydrogenase and lactate dehydrogenase.
The mitochondrial volume densities (V mit) of the different fiber types (type 1, type 2A, type 2B) were estimated in bilaterally obtained biopsies from 22 patients with unilateral intermittent claudication. These data, which were obtained from structurally intact fibers, were compared with clinical data from the same subject In both the asymptomatic and symptomatic legs, V mit 1 > V mit 2A > V mit 2B. Furthermore, V mit 1 covariated with Vmit 2A and V mit 2A with V mit 2B in the asymptomatic legs (as in healthy subjects) but not in the symptomatic legs. V mit 2 (mainly Vmit 2A) covariated with the age of the subjects in both legs. V mit Tot was higher in the symptomatic legs than in the asymptomatic legs. This was mainly due to increase in the oxidative fibers, type 1 and type 2A. Usually, V mit in the asymptomatic legs covariated sig-nificantly with the results of the functional tests (initial pain and maximum walking tolerance), while only V mit 2A in the symptomatic legs showed such a corre lation. However, the difference between the two legs concerning V mit 1 was also correlated to the walking tolerance. Patients with high stenosis or occlusion showed higher Vmit Tot than did those with low obstacles. The results conclusively show that a fiber type-specific adaptation to ischemia occurs through an increase of mitochondrial content of oxidative fibers, which suggests that hypoxia may influence the control of synthesis or degradation of mitochondrial proteins.
Muscle fiber fine structure was quantitatively analyzed in 70 longitudinally and 65 transversely sectioned fibers from 10 control subjects without weakness. The average mitochondrial fraction of the fiber volume is close to 4 percent and the mean size of a mitochondrion is about 0.1 mum2. The sarcotubular surface area per unit fiber volume is close to 1.5 mum2/mum3 in transverse sections and 0.65 times this value in longitudinal sections. Only one-third of all fibers contain lipid droplets in the sectioned plane, and for all fibers the droplets account for approximately 0.12 percent of the fiber volume. Variations with the age and sex of the subjects and with different muscles were analyzed and the feasibility of typing human muscle fibers at the ultrastructural level was evaluated.
A morphological and quantitative study of skeletal muscle fibres and of capillary supply was performed on needle and open biopsies of quadriceps femoris muscle from 40 patients with chronic progressive external ophthalmoplegia (CPEO) with partial deficiency of cytochrome c oxidase (COX). Muscle biopsies from 5 healthy adult subjects were used as control. The study was carried out by light and electron microscopy, using an Automatic Interactive Image Analysis System (IBAS I,II). A significant decrease in fibre diameters and preferential type I fibre atrophy was seen. Red ragged fibres and fibres with cytoarchitectural changes after enzyme-histochemical reactions for detection of oxidative activities were also observed. Seventy per cent of affected fibres showed an intense subsarcolemmal rim of oxidative activity corresponding to ultrastructural accumulation of enlarged, polymorphous mitochondria in subsarcolemmal areas. The study of the capillary distribution in muscle revealed a reduction of the number of capillaries per fibre and surrounding a fibre. The primary metabolic error of the disease with defects in the oxygen utilisation, the fibre atrophy and the muscle disuse are the possible variables influencing the capillary number in CPEO patients.
It is well documented in both animal and human studies that unaccustomed, particularly eccentric, muscle exercise may cause damage of muscle fiber contractile and cytoskeletal components. These injuries typically include: Z-band streaming and dissolution, A-band disruption, disintegration of the intermediate filament system, and misalignment of the myofibrils. The mechanical basis for this damage is suggested to be due to the fiber strain magnitude rather than the absolute stress imposed on the fiber. We hypothesize that eccentric contraction-induced damage occurs early in the treatment period, i.e., within the first few minutes. The structural abnormalities predominate in the fast-twitch glycolytic fibers. In the final section of this paper, we hypothesize a damage scheme, based on the muscle fiber oxidative capacity as a determining factor.
In response to patterns of chronic electrical stimulation that increase its overall level of use, mammalian skeletal muscle becomes highly resistant to fatigue. The metabolic basis for this adaptation is well documented in the rabbit, but up to now it has not been possible to identify analogous changes in the dog. In this study, canine latissimus dorsi muscles were stimulated in situ for 2, 6 and 12 mo. Marked increases in fatigue resistance were consistently demonstrated. Citrate synthase and succinic dehydrogenase, conventionally used as markers of oxidative metabolism, did not increase in activity, but enzymes involved in major pathways supplying substrates to the tricarboxylic acid cycle increased up to threefold. Stimulation elevated the volume fraction of mitochondria 1.5-fold and that of lipid droplets 4.5-fold. After 6 mo of stimulation, mean fiber diameter had decreased by 30% and the area occupied by nonmuscle tissue had increased by 11%; these changes showed no further progression at 12 mo. Thus stimulated muscle becomes stably adapted to an increase in use, but the metabolic strategies for achieving increased fatigue resistance vary between species.
Plasma creatine kinase (CK) activity has often been used as a marker of exercise-induced skeletal muscle damage. While the pattern of muscle damage following eccentric exercise has been established in young adults, there is little data available on eccentric exercise-induced muscle damage in older individuals. The purpose of this study was to compare ultrastructural changes in skeletal muscle following high intensity eccentric exercise of young and older men and to determine whether CK activity is a reliable predictor of muscle damage. Five young (20-30 yr) and five older untrained men (59-63 yr) performed three 15-min bouts of eccentric exercise at 90, 80, and 70% of maximal concentric power output. There was a prolonged increase in CK up to 10 d following exercise that was not significantly different between groups. Light and electron microscopic examination of needle biopsies obtained from the vastus lateralis showed evidence of focal damage in greater than 90% of the post-exercise fibers examined in the older subjects, compared with values ranging from 5 to 50% reported previously in young subjects. Quantitative analysis using light microscopy showed greater damage in the older subjects than reported previously in young subjects. These data suggest that older adults experience greater muscle damage following eccentric exercise than young subjects, which may be due in part to the smaller muscle mass and lower VO2max seen in older men. In addition, there was no relationship between CK activity and the corresponding amount of muscle damage observed in each subject, suggesting that CK activity may be a poor predictor of exercise-induced muscle damage.
A morphometric analysis was made of nuclei and cytoplasmic structures in electron micrographs of chondrocytes in the non-calcified layer of articular cartilage of the femoral condyles in adult mouse and dog and of the human incus. Mitochondrial volume density (% cytoplasm) was lower in dog than in mouse cells or cells of the incus. It was also lower in the cells of deep zone cartilage than in superficial cells. Analysis of keratocytes of the corneal stroma in mouse and sheep gave similar findings to that in cartilage. Mitochondrial volume density was higher in mouse than in sheep keratocytes and, in sheep, higher in subepithelial (anterior) cells than in cells of the intermediate and subendothelial strata. Values in human stromal cells were similar to those in the sheep. Endoplasmic reticulum volume density was higher in mouse than in sheep keratocytes. Lipid and filaments were more abundant in dog than in mouse chondrocytes but keratocytes contained little or none. Mitochondrial volume densities correlate with diffusion distances (tissue thickness) from the sources of nutrition, for example, for oxygen, in the two tissues. The relationship to certain aspects of chondroitin sulphate and keratansulphate synthesis and topographical distribution in cartilage and cornea is discussed.