Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

Summary The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semi-thin (0.25 and 1 m) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for lightmicroscopic analysis may account for the differences.The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.     

Light microscopic visualization of the reaction product of cerium used for localization of peroxisomal oxidases

The reaction product of cerium used for localization of peroxisomal oxidases is highly electron-dense but lacks sufficient contrast at the light microscopic level. We describe two methods for converting the reaction product of cerium to colored compounds visible by light microscopy. The first method is based on 3,3'-diaminobenzidine (DAB) amplification of transition metal compounds, of which cerium is one. Sections of glutaraldehyde-fixed rat liver or kidney are incubated first in media for various oxidases containing CeCl3, followed by treatment with DAB in Na acetate buffer, pH 5.3. To prevent any interference by the peroxidatic activity of catalase, NaN3 or Na pyruvate is added to the DAB amplification medium. Staining with DAB can be further intensified with NiCl2 or CoCl2. The second method is based on the conversion of the cerium reaction product with alkaline lead citrate and the final visualization of the lead compound with ammonium sulfide. These methods allow the evaluation of large sections for peroxisomal oxidases by light microscopy, making close correlation between light and electron microscopy possible.     

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

Summary The feasibility of the application of an electronic image analyzer, the Texture Analysis System (TAS) (Leitz Wetzlar, FRG), for fast automatic ultrastructural morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained selectively with the alkaline DAB procedure for localization of catalase in order to obtain sufficient contrast for automatic detection by TAS. Electron micrographs of ultrathin sections from this material were analyzed both automatically by TAS and manually by using a digitizer tablet connected with an Apple IIe microcomputer. The results showed negligible differences. As far as the speed of the operation is concerned, the image analysis was 4–5 times faster than the manual technique. In further studies, the importance of using DAB-stained sections for accurate morphometric studies of peroxisomes was demonstrated by comparing the results of such DAB-stained preparations with unstained material. This revealed that the numerical density was lower and the average profile diameter higher in unstained sections. The value for volume density was also affected, being about 30% lower in such preparations. It is likely that in unstained preparations small peroxisomes without crystalline nucleoids were frequently not identified as such and were not taken into account in morphometric calculations.These observations establish that computer-controlled electronic image analysis in conjunction with selective cytochemical staining of peroxisomes for catalase provides a fast, accurate and reliable method for ultrastructural morphometric studies of this organelle in rat liver.     

Zonal heterogeneity of peroxisome proliferation and morphology in rat liver after gemfibrozil treatment

The effect of gemfibrozil on the fine structure of peroxisomes across the rat liver lobule was investigated by light and electron microscopy using the alkaline diaminobenzidine (DAB) medium for the visualization of catalase peroxidatic activity. The oral administration of gemfibrozil for 2 weeks induces a striking heterogeneity in the lobular distribution of peroxisomes. The size and shape of peroxisomes, variety of matrix modifications, catalase content, and position within the cell, are functions of the zonal localization of the hepatocytes. The largest and most numerous peroxisomes were found in the centrilobular region indicating that these cells are most sensitive to peroxisome proliferation. On the other hand, the greatest variety of peroxisome shapes and matrix alterations (tubules and plates) was seen more peripherally in the mid-zonal and periportal regions. The larger, round centrilobular peroxisomes stained less intensely than the elongated peroxisomes found more peripherally, indicating a discrepancy between peroxisome size and catalase content. A distinct population of small irregularly shaped peroxisomes, lacking matrix specializations and containing variable catalase content, was found in the mid-zonal region. Peroxisomes in the centrilobular region were located within areas of the cell containing SER and glycogen while those in the more peripheral region were relegated to areas of the cytoplasm separate from RER and SER. In addition to modifications of peroxisomes, gemfibrozil treatment resulted in a proliferation and formation of whorled configurations of SER. This was particularly evident in the mid-zonal region, where single peroxisomal profiles could be seen surrounded by whorls of SER membranes. The results suggest that rat liver hepatocytes of the centrilobular region are the most sensitive to peroxisome proliferation and those of the periportal area are most susceptible to peroxisome matrix alterations after gemfibrozil treatment.     

Nafenopin-induced proliferation of peroxisomes in the small intestine of mice.

The effect of nafenopin on the epithelial cells of the small intestine of mice was studied. After 17 days the control and nafenopin-treated groups were sacrificed. The tissues were incubated in alkaline DAB medium. Ultra-thin sections of small intestinal tissue from both groups were examined by electron microscopy. Electron micrographs were prepared and examined stereologically so that any morphologic differences in the epithelial cell peroxisomes and mitochondria between the experimental and control groups could be evaluated quantitatively. In the nafenopin-treated group proliferation of peroxisomes occurred, as indicated by significant increases in volume, and surface and numerical density of these structures compared with controls. No such alterations were found in the mitochondria. Our results show that the response of small intestinal epithelial cells to nafenopin is analogous to that produced in hepatocytes by the same drug. Hepatocyte peroxisomes are supposed to be involved in lipid metabolism and it seems that small intestinal epithelial peroxisomes play a similar role.     

Peroxisomes: 40 years of histochemical staining,personal reminiscences

The historical circumstances that led to the discovery of the 3,3′-diamino-benzidine (DAB) method for staining of peroxisomes 40 years ago are reviewed. In the course of studies on the uptake and absorption of horse radish peroxidase in mammalian liver, in sections incubated for detection of peroxidase activity in DAB, it was noted that peroxisomes also stained positively for peroxidase activity. Subsequently, it was revealed that the peroxidatic activity of catalase, which is abundantly present in peroxisomes, is responsible for that staining. This notion was confirmed in quantitative biochemical studies with crystalline beef liver catalase and in tracer studies using catalase as an ultrastructural tracer. The application of the DAB method led to the discovery of peroxisomes as a ubiquitous eukaryotic cell organelle, attracting great interest in their investigation in biomedical research.     

Peroxisomes of the rat cardiac and soleus muscles increase after starvation. A biochemical and immunocytochemical study

We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 microns 2 of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/micron 2) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.     

Peroxisomes in digestive gland cells of the mussel Mytilus galloprovincialis Lmk. Biochemical, ultrastructural and immunocytochemical characterization.

The present study was undertaken because of the paucity of information on peroxisomes in molluscs and the increasing importance of these organisms as sensitive indicators of environmental pollution. Peroxisomes were identified by electron microscopy in all three main cell types of the digestive gland of the bivalve mollusc Mytilus galloprovincialis Lmk. They stained weakly with the alkaline diaminobenzidine reaction but showed distinct immunolabeling with an antibody against mammalian catalase by the postembedding protein A-gold procedure. In addition, mussel digestive gland peroxisomes were isolated by differential and metrizamide-density gradient centrifugation, and a 30-fold enrichment of catalase and a 20-fold enrichment of palmitoyl-CoA oxidase was obtained over the initial homogenate. By Western blotting, isolated peroxisomes crossreacted with antibodies to catalase and, furthermore, specific and prominent labeling of isolated peroxisomes was also demonstrated in thin sections incubated with anti-catalase antibodies. These observations establish that peroxisomes in molluscan digestive gland contain the peroxisomal marker enzymes catalase and acyl-CoA oxidase and that they can be labeled by cytochemical and immunocytochemical techniques. Further studies of alterations of molluscan peroxisomes by environmentally relevant xenobiotics are warranted.     

Mammalian SOD2 is exclusively located in mitochondria and not present in peroxisomes

Superoxide dismutases (SODs) are metalloenzymes that belong to the essential antioxidant enzyme systems of virtually all oxygen-respiring organisms. SODs catalyze the dismutation of highly reactive superoxide radicals into hydrogen peroxide and molecular oxygen. For the subcellular localization of the manganese superoxide dismutase (SOD2) in eukaryotic cells, a dual mitochondrial localization and peroxisomal localization were proposed in the literature. However, our own observation from immunofluorescence preparations of human and mouse tissues suggested that SOD2 serves as an excellent marker protein for mitochondria but never co-localized with peroxisomes. To clarify whether our observations were correct, we have carefully reinvestigated the subcellular localization of SOD2 using sensitive double-immunofluorescence methods on frozen and paraffin sections as well as in cell culture preparations. In addition, ultrastructural analyses were performed with post-embedding immunoelectron microscopy on LR White sections as well as labeling of ultrathin cryosections with various immunogold techniques. In all morphological experiments, the SOD2 localization was compared to one of the catalase, a typical marker protein for peroxisomes, solely localized in these organelles. Moreover, biochemical subcellular fractions of mouse liver was used to isolate enriched organelles and highly purified peroxisomal fractions for Western blot analyses of the exact subcellular distributions of SOD2 and catalase. All results with the various methodologies, tissues, and cell types used revealed that catalase and SOD2 were always confined to distinct and separate subcellular compartments. SOD2 was unequivocally in mitochondria, but never present in peroxisomes. Furthermore, our results are supported by accumulating database information on organelle proteomes that also indicate that SOD2 is a pure mitochondrial protein.     

Cytochemistry of human catalase. The demonstration of hepatic and renal peroxisomes by a high temperature procedure.

The cytochemical demonstration of marker enzymes for subcellular organelles permits light microscopic analysis of their structure and function in normal and diseased tissues. Currently available staining procedures for the peroxidatic activity of catalase in peroxisomes of plant and animal cells yield weak and inconsistent light microscopic staining when applied to human tissues. We have developed a simple and sensitive high temperature procedure that clearly and reproducibly stains these abundant, but poorly understood, organelles in biopsy specimens of human liver and kidney. This method utilizes formaldehyde fixation, a modified diaminobenzidine (DAB) medium, incubation at 45 degrees C and postosmication for both light and electron microscopy.     

Diaminobenzidine as a Myelin Stain in Semithin Plastic Sections

A diaminobenzidine (DAB) stain for myelin in glutaraldehyde fixed, osmicated, semithin epoxy sections is described. One or 1.5 μm sections, dried onto slides, are first etched with a 1:2 dilution of saturated sodium ethox-ide:absolute ethanol, then incubated in 0.05% aqueous DAB with 0.01% hydrogen peroxide. DAB specifically stains osmium fixed myelinated nerve fibers. This permits high resolution light microscopic study of myelinated nerve fibers in semithin sections of tissues that also can be studied by electron microscopy.     

Peroxisomes of the rat cardiac and soleus muscles increase after starvation

Summary We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 m² of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/m²) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.     

Cytochemical detection of catalase with 3,3'-diaminobenzidine. A quantitative reinvestigation of the optimal conditions

The influence of various parameters of fixation and incubation upon the oxidation of DAB by catalase have been analyzed. Crystalline beef liver catalase was fixed with different concentrations of glutaraldehyde and peroxidatic activity was determined spectrophotometrically using DAB as hydrogen donor. Although aldehyde fixation appeared to be important in elicitation of the peroxidatic activity of catalase, the final pigment production after 60 min incubation was optimal with the lowest concentration of glutaraldehyde (1%), after the shortest fixation period (30 min), and at the lowest temperature (5 degrees C) tested. Similarly cytochemical studies with rat kidney sections incubated for 10 min confirmed that the staining of peroxisomes in proximal tubules was strongest after the "mildest" fixation conditions. The pH and the temperature of incubation were closely interrelated, so that at room temperature (25 degrees C) the maximal pigment production was obtained at pH 10.5, but incubation at 45 degrees C gave the strongest staining at pH 8.5. The production of pigment increased with higher DAB concentrations which required larger amounts of H2O2 in the incubation medium. Cytochemical studies on renal peroxisomes were in agreement with these biochemical findings. The observations indicate that there are several options for the localization of catalase depending on the fixation and incubation conditions. Hence, these conditions should be selected according to the tissue and the purpose of the study. Examples for such selective applications are presented.     

Specific cytochemical stains in the image analysis of subcellular organelles

This paper describes our work concerning densitometry and morphometry of subcellular structures in thin sections. The techniques of automatic image analysis were applied to light and electron microscopic observations of enzymatically stained lysosomes, renal brush borders and mitochondria (in human and rat kidney) and peroxisomes (in human liver). To obtain significant measurements of the enzymatic activity, specific staining techniques were developed and applied, including an improved staining of acid phosphatase for lysosomes. Optical densities were obtained by videodensitometry and electron densities of peroxisomes were obtained by digitizing and processing scanning transmission electron microscopic images. In subsequent steps, delineations and parameter estimation are performed by software. Included was an examination of delineation techniques, which showed improved results from the use of a newly developed local boundary search algorithm. The combination of these techniques was used to study changes in peroxisome and lysosome compartment in liver and kidney, some results of which are also reported.     

Selective cytochemical localization of peroxidase, cytochrome oxidase and catalase in rat liver with 3,3'-diaminobenzidine

In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specificity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15-30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylte buffer pH 6.5 and 0.02% H2O2. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) buffer at pH 10.5 and 0.15% H2O2. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Selective cytochemical localization of peroxidase,cytochrome oxidase and catalase in rat liver with 3,3′-diaminobenzidine

Summary In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H₂O₂. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H₂O₂. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Cytochemical localization of catalase in leaf microbodies (peroxisomes)

Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3''-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.     

The role of 15-lipoxygenase in disruption of the peroxisomal membrane and in programmed degradation of peroxisomes in normal rat liver.

Our earlier electron microscopic observations revealed that prolonged exposure of glutaraldehyde-fixed rat liver sections to buffer solutions induced focal membrane disruptions of peroxisomes with catalase diffusion as shown cytochemically. Recently, it was suggested that 15-lipoxygenase (15-LOX) might be involved in natural degradation of membrane-bound organelles in reticulocytes by integrating into and permeabilizing the organelle membranes, leading to the release of matrix proteins. We have now investigated the localization of 15-LOX and its role in degradation of peroxisomal membranes in rat liver. Aldehyde-fixed liver slices were incubated in a medium that conserved the 15-LOX activity, consisting of 50 mM HEPES-KOH buffer (pH 7.4), 5 mM mercaptoethanol, 1 mM MgCl(2), 15 mM NaN(3), and 0.2 M sucrose, in presence or absence of 0.5-0.05 mM propyl gallate or esculetin, two inhibitors of 15-LOX. The exposure of aldehyde-fixed liver sections to this medium induced focal disruptions of peroxisome membranes and catalase diffusion around some but not all peroxisomes. This was significantly reduced by both 15-LOX inhibitors, propyl gallate and esculetin, with the latter being more effective. Double immunofluorescent staining for 15-LOX and catalase revealed that 15-LOX was co-localized with catalase in some but not all peroxisomes in rat hepatocytes. By postembedding immunoelectron microscopy, gold labeling was localized on membranes of some peroxisomes. These observations suggest that 15-LOX is involved in degradation of peroxisomal membranes and might have a physiological role in programmed degradation and turnover of peroxisomes in hepatocytes. (J Histochem Cytochem 49:613-621, 2001)     

Peroxisomes in sebaceous glands. IV. Aggregates of tubular peroxisomes in the mouse Meibomian gland

The occurrence of peroxisomes, their morphogenesis during the process of sebaceous transformation and their spatial relationship to the endoplasmic reticulum and lipid droplets were investigated by light and electron microscopy after visualization of the peroxidatic activity of catalase using an alkaline diaminobenzidine medium. The morphological alterations of peroxisomes display a characteristic sequence: During cellular differentiation, a remarkable proliferation of exclusively tubular, diaminobenzidine-reactive peroxisomes occurs. As maturation proceeds, an extensive elongation of tubular peroxisomes is seen. Concomitantly, they are densely packed in a regular, hexagonal arrangement and both the diameter and the catalase content gradually decreases. The most conspicuous feature of mature glandular cells are numerous highly organized aggregates of tubular, almost unstained peroxisomes with a diameter of 50 nm, arranged in a hexagonal pattern. They resemble adjacent tubular profiles of smooth endoplasmic reticulum. However, membrane continuities between these two compartments were never observed. During lethal disintegration peroxisomes subsequently decrease in number, probably by rapid sequestration within autophagolysosomes. The role of tubular peroxisomes in the biosynthesis of wax esters in the mouse Meibomian gland is discussed.     

Three-dimensional ultrastructural analysis of peroxisomes in HepG2 cells

Peroxisomes in the human hepatoblastoma cell line, HepG2, exhibit distinct alterations of shape, size, and distribution, dependent on culture conditions (cell density, duration in culture, and presence of specific growth factors). Although many cells with elongated tubular peroxisomes are present in thinly seeded cultures, spherical particles forming large focal clusters are found in confluent cultures. The authors have analyzed the ultrastructure and the spatial relationship of peroxisomes of HepG2 cells at different stages of differentiation, using three-dimensional (3D)-reconstruction of ultrathin serial sections, and electronic image processing. Cells were prepared for immunofluorescence using different antibodies against peroxisomal matrix and membrane proteins, as well as for electron microscopy after the alkaline 3,3′-diaminobenzidine staining for catalase. The results indicate that the tubular peroxisomes, which can reach a length of several microns, are consistently isolated, and never form an interconnected peroxisomal reticulum. At the time of disappearance of tubular peroxisomes, rows of spherical peroxisomes, arranged like beads on a string, are observed, suggesting fission of tubular ones. In differentiated confluent cultures, clusters of several peroxisomes are seen, which, by immunofluorescence, appear as large aggregates, but after 3D reconstruction consist of single spherical and angular peroxisomes without interconnections. The majority of such mature spherical peroxisomes (but not the tubular ones) exhibit tail-like, small tubular and vesicular attachments to their surface, suggesting a close functional interaction with neighboring organelles, particularly the endoplasmic reticulum, which is often observed in close vicinity of such peroxisomes.