3D microscopic imaging and evaluation of tubular tissue architecture.

3D microscopy and image analysis provide reliable measurements of length, branching, density, tortuosity and orientation of tubular structures in biological samples. We present a survey of methods for analysis of large samples by measurement of local differences in geometrical characteristics. The methods are demonstrated on the structure of the capillary bed in a rat brain.

Yet another decennium of the Institute of Physiology: a dynamic interplay of innovative approaches and 60 years of tradition.

I am pleased to introduce this special issue of Physiological Research published on the occasion of the 60th anniversary of the Institute of Physiology. It is only a second issue of this kind, the previous one being Physiological Research 53 (Suppl. 1) 2004. Since then, the Institute contributed its expertise to modern fields of physiology such as cardiovascular physiology, neurophysiology, energy metabolism, membrane transport, chronobiology, as well as relevant methodology. Diverse local and international collaboration has augmented such effort, as summarized in the attached Synopsis outlining the most significant achievements of Institute's departments during the past ten years. I very much hope that achievements of this kind will become Institute's tradition justifying at least equally optimistic forthcoming special issues in the decades to come.

The branching pattern of villous capillaries and structural changes of placental terminal villi in type 1 diabetes mellitus.

Maternal diabetes is associated with changes of the placental structure. These changes include great variability of vascularity manifested by strikingly hypovascular as well as hypervascular terminal villi. In this paper, normal placental terminal villi and pathological villi of type 1 diabetic placentas were compared concerning the structure of villous stroma, spatial arrangement of villous capillary bed and quantitative assessment of capillary branching pattern. Formalin fixed and paraffin embedded specimens of 14 normal and 17 Type 1 diabetic term placentas were used for picrosirius staining, vimentin and desmin immunohistochemistry and confocal microscopy. 3D models of villi and villous capillaries were constructed from stacks of confocal optical sections. Hypervascular as well as hypovascular villi of diabetic placenta displayed changed structure of villous stroma, i.e. the collagen envelope around capillaries looked thinner and the network of collagen fibers seemed less dense. The desmin immunocytochemistry has shown that stromal cells of hypervascular as well as hypovascular villi appeared nearly or completely void of desmin filaments. In comparison with normal villi, capillaries of hypovascular villi had a smaller diameter and displayed a markedly wavy course whereas in hypervascular villi numerous capillaries occurred in reduced stroma and often had a large diameter. The quantitative assessment of capillary branching has shown that villous capillaries are more branched in diabetic placentas. It is concluded that type 1 maternal diabetes enhances the surface area of the capillary wall by elongation, enlargement of diameter and higher branching of villous capillaries and disrupts the stromal structure of terminal villi.

Characterization of the capillary network in skeletal muscles from 3D data.

In this review we present immunohistochemical methods for visualization of capillaries and muscle fibres in thick muscle sections. Special attention is paid to the procedures that preserve good morphology. Applying confocal microscopy and virtual 3D stereological grids, or tracing of capillaries in virtual reality, length of capillaries within a muscle volume or length of capillaries adjacent to a muscle fibre per fibre length, fibre surface area or fibre volume can be evaluated by an unbiased approach. Moreover, 3D models of capillaries and muscle fibres can be produced. Comparison of the developed methods with counting capillary profiles from 2D sections is discussed and the reader is warned that counting capillary profiles from 2D sections can underestimate the capillary length by as much as 75 percent. Application of the described 3D methodology is illustrated by the anatomical remodelling of capillarity during acute denervation and early reinnervation in the rat soleus and extensor digitorum longus muscles.

Software for muscle fibre type classification and analysis.

Fibre type determination requires a large series of differently stained muscle sections. The manual identification of individual fibres through the series is tedious and time consuming. This paper presents a software that enables (i) adjusting the position of individual fibres through a series of differently stained sections (image registration) and identification of individual fibres through the series as well as (ii) muscle fibre classification and (iii) quantitative analysis. The data output of the system is the following: numerical and areal proportions of fibre types, fibre type size and optical density (grey level) of the final reaction product in every fibre. The muscle fibre type can be determined stepwise, based on one set of stained sections while further, newly stained sections can be added to the already defined muscle fibre profile. Several advantages of the presented software application in skeletal muscle research are presented. The system is semiquantitative, flexible, and user friendly.

Software for muscle fibre type classification and analysis.

Fibre type determination requires a large series of differently stained muscle sections. The manual identification of individual fibres through the series is tedious and time consuming. This paper presents a software that enables (i) adjusting the position of individual fibres through a series of differently stained sections (image registration) and identification of individual fibres through the series as well as (ii) muscle fibre classification and (iii) quantitative analysis. The data output of the system is the following: numerical and areal proportions of fibre types, fibre type size and optical density (grey level) of the final reaction product in every fibre. The muscle fibre type can be determined stepwise, based on one set of stained sections while further, newly stained sections can be added to the already defined muscle fibre profile. Several advantages of the presented software application in skeletal muscle research are presented. The system is semiquantitative, flexible, and user friendly.

Advantages and pitfalls of using free-hand sections of frozen needles for three-dimensional analysis of mesophyll by stereology and confocal microscopy.

The anatomical structure of mesophyll tissue in the leaf is tightly connected with many physiological processes in plants. One of the most important mesophyll parameters related to photosynthesis is the internal leaf surface area, i.e. the surface area of mesophyll cell walls exposed to intercellular spaces. An efficient design-based stereological method can be applied for estimation of this parameter, using software-randomized virtual fakir test probes in stacks of optical sections acquired by a confocal microscope within thick physical free-hand sections (i.e. acquired using a hand microtome), as we have shown in the case of fresh Norway spruce needles recently. However, for wider practical use in plant ecophysiology, a suitable form of sample storage and other possible technical constraints of this methodology need to be checked. We tested the effect of freezing conifer needles on their anatomical structure as well as the effect of possible deformations due to the cutting of unembedded material by a hand microtome, which can result in distortions of cutting surfaces. In the present study we found a higher proportion of intercellular spaces in mesophyll in regions near to the surface of a physical section, which means that the measurements should be restricted only to the middle region of the optical section series. On the other hand, the proportion of intercellular spaces in mesophyll as well as the internal needle surface density in mesophyll did not show significant difference between fresh and frozen needles; therefore, we conclude that freezing represents a suitable form of storage of sampled material for proposed stereological evaluation.

Three-dimensional arrangement of the capillary bed and its relationship to microrheology in the terminal villi of normal term placenta.

Spatial arrangement of the capillary bed, manifestations of its growth and symmetry of capillary branching were studied in peripheral villi of normal human placenta at term using confocal microscopy and image analysis. Unlike the model that has been accepted so far, it was shown that the arrangement of the capillary bed in terminal villi varied from simple, U-like loops to a richly branched network. Three different categories of terminal villi (TV) were recognised: Signs of capillary elongation and sprouting were observed in the villous capillary bed. Based on the assessment of the mean cross-sectional areas of capillaries constituting simple, Y-like capillary bifurcation in terminal villi, the capillary branching was found to be asymmetric. Therefore, we conclude that the conditions for the "plasma skimming" effect are met in human placenta.

Quantitative analysis of embryonic kidney impairment by confocal microscopy and stereology: effect of 1,2-dibromoethane in the chick mesonephros.

1. Chick embryos in ovo were treated with a teratogenic dose of 1,2-dibromoethane (DBE) on embryonic day (ED) 3. On ED 6 and 10, histological sections of whole embryos were prepared for confocal microscopy. In parallel, mesonephroi of 10-d-old embryos were dissected for in situ staining with acridine orange (AO), a fluorescence probe for lysosomes. 2. DBE impaired differentiation of renal vessels which manifested as a delay in rearrangement of primitive renal vascular architecture on ED 6 and a significant reduction of the mesonephric vascularisation on ED 10. This was accompanied by delayed functional maturation of embryonic kidney, as suggested by staining with AO. 3. Renal vessels appeared to be more susceptible to DBE than tubules. Unequal growth of these renal components might be a cause of DBE-induced spatial disorganisation of tubular apparatus. 4. Nephrotoxic effects of DBE during the embryonic period are associated primarily with damage to the renal blood supply. 5. Confocal microscopy, stereological methods and three-dimensional reconstruction of developing tissues are useful tools to investigate pathogenic processes during embryonic development.

Improvement in volume estimation from confocal sections after image deconvolution.

The confocal microscope can image a specimen in its natural environment forming a 3D image of the whole structure by scanning it and collecting light through a small aperture (pinhole), allowing in vivo and in vitro observations. So far, the confocal fluorescence microscope (CFM) is considered a true volume imager because of the role of the pinhole that rejects information coming from out-of-focus planes. Unfortunately, intrinsic imaging properties of the optical scheme presently employed yield a corrupted image that can hamper quantitative analysis of successive image planes. By a post-image collection restoration, it is possible to obtain an estimate, with respect to a given optimization criterium, of the true object, utilizing the impulse response of system or Point Spread Function (PSF). The PSF can be measured or predicted so as to have a mathematical and physical model of the image-formation process. Further modelling and recording noise as an additive Gaussian process has used the regularized Iterative Constrained Tykhonov Miller (ICTM) restoration algorithm for solving the inverse problem. This algorithm finds the best estimate iteratively searching among the possible positive solutions; in the Fourier domain, such an approach is relatively fast and elegant. In order to compare the effective improvement in the quantitative image information analysis, we measured the volume of reference objects before and after image restoration, using the isotropic Fakir method.

A novel staining method for quantification and 3D visualisation of capillaries and muscle fibres.

The aim of this study was to introduce a combined fluorescent staining that clearly demonstrates capillaries and distinguishes them from the basal lamina of muscle fibres in skeletal muscle tissue. The triple staining with CD31, Griffonia (Bandeira) simplicifolia lectin (GSL I) and laminin efficiently distinguishes vascular endothelium from the basal lamina of skeletal muscle fibres in physiological and pathological conditions. The presented triple staining method has several advantages, which facilitate quantitative analysis of the capillary network, and its relation to individual muscle fibres.

Confocal stereology and image analysis: methods for estimating geometrical characteristics of cells and tissues from three-dimensional confocal images.

A short review of confocal stereology and three-dimensional image analysis is presented, pointing out the achievements accomplished in this field by the Department of Biomathematics (Institute of Physiology, Prague). One of the methods of confocal stereology, the fakir method for surface area estimation, developed by this laboratory, is described. Methods for automatic measurement of geometrical characteristics of microscopical structures, based on 3-D image processing or surface triangulation, are discussed and compared with interactive stereological methods. Three-dimensional reconstruction programs and software implementation of stereological and digital methods as well as their practical applications are presented. The future trends are discussed.

Confocal microscopy and stereology: estimating volume, number, surface area and length by virtual test probes applied to three-dimensional images.

The opportunities of confocal microscopy applied to morphometry of microscopical structures are presented and demonstrated on stereological methods based on evaluation of optical sections within a thick slice and using computer-generated virtual test probes. Such methods, allowing arbitrary orientation of the thick slice, can be used for estimating volume, number, surface area, and length. The methods using spatial grid of points, disector, fakir, and slicer probes are described and illustrated by different examples using our freeware 3DTOOLS software and their variance and applicability are discussed. It is shown that shifted triple or quadruple spatial grids of lines are very efficient for the surface area and volume estimation by the fakir method.

Three-dimensional study of the capillary supply of skeletal muscle fibres using confocal microscopy.

Three-dimensional (3D) study of capillary network of individual muscle fibres in rat extensor digitorum longus (EDL) and soleus (SOL) muscles is presented. Stereology and 3D reconstruction techniques were applied to stacks of serial optical sections recorded by a confocal microscope from thick muscle slices. The results suggest that SOL muscle fibres have a larger surface area and volume as well as a larger length of capillaries per fibre length than EDL. On the other hand, these two muscles have a similar ratio of capillary length to fibre surface area. The 3D approach to evaluation of muscle fibre capillarization brings many advantages over traditional measurements made on single muscle sections and could also be applied to the study of angiogenesis in other tissues.

STESYS2: extended STESYS software for MS Windows.

The STESYS2 software is a new version of the IBM PC software supporting interactive stereological measurements. In comparison with the previous STESYS, it is enhanced by a number of useful options, e.g. on-line image input via a TV camera coupled with a microscope operating under MS Windows OS. The main advantage, when compared with other such software packages, is the design of the STESYS2 as a module of the freeware image processing system Image Tool which provides a user-friendly environment including a number of image processing and preprocessing routines. Capabilities of the STESYS2 are illustrated by a practical example: estimation of the surface area of capillaries in the terminal villi of human placenta by the Sandau spatial grid method.

Comparison of several digital and stereological methods for estimating surface area and volume of cells studied by confocal microscopy.

BACKGROUND: The implementation of different methods for estimating the surface area and volume of cells studied by confocal microscopy was developed. The methods were compared from the point of view of their precision, applicability and efficiency. METHODS: Interactive stereological methods (spatial grid method, fakir method, Cavalieri principle) as well as automatic digital methods (digital Crofton method, voxel counting, triangulation method, iso-intensity contouring method) were considered. The methods were tested on model geometrical solids and on real volume images consisting of a stack of serial sections encompassing entire tobacco BY-2 cells or cell chains. RESULTS: It is shown that many of the studied methods are very precise when applied to cells of simple or moderately complex shapes. The automatic digital methods are fast and precise but their applicability is limited by the necessity to segment automatically the object surface and to find an optimal resolution. This limitation is not present in stereological methods which are applied interactively and thus are more time-consuming. CONCLUSIONS: The presented implementations of the fakir method and the Cavalieri principle enable interactive, unbiased and efficient estimation of the cell surface area and volume. The recommended steps for measuring the surface area and/or volume of objects studied by confocal microscopy are described.

STESYS software for computer-assisted stereology.

A software system STESYS for interactive and flexible generation of stereological test systems is described. STESYS enables to implement many of the recent unbiased stereological methods applied to biomedical research and clinical diagnosis by using a simple personal computer. Advantages of the STESYS software are illustrated by several examples of stereological measurements for estimating the number, total and mean cross-sectional area, volume and surface area.

Estimating surface area by the isotropic fakir method from thick slices cut in an arbitrary direction.

The proposed fakir method for estimating surface area is based on counting the intersections between the surface lying within a thick slice, and an isotropic spatial grid consisting of a combination of linear probes called fakir probes. An unbiased procedure using a directly randomized spatial grid rather than sections with randomized directions is presented. The method is applicable if perfectly registered serial sections of the surface are available in a thick slice while the direction of the slice can be arbitrary. The efficiency of the fakir method using different arrangements of orthogonal triplets of fakir probes is evaluated and it is shown that mutually shifted probes are superior to non-shifted ones. The application software for interactive counting of intersections between computer-generated fakir probes and the surface within the stack of digitized images is described and demonstrated by two examples: estimation of the surface area of individual tobacco cell chains using a confocal microscope, and estimation of the total area of exposed surface of mesophyll cells in a barley leaf using a wide-field transmission microscope.

Hyperthermia in the chick embryo: HSP and possible mechanisms of developmental defects.

Although hyperthermia is an established teratogen in all species studied and the cellular heat shock response is well known, the mechanisms of developmental deviation remain obscure. We have used a chick model system in which fertilized eggs containing embryos at presomite and/or early somite stages (HH 4-10) were exposed to 45 degrees C for 180 min. Six hours following treatment we did not observe any overt morphological disturbance, but at twelve hours following exposure (when controls reached HH 11-13) embryos exposed at late streak stages (HH 4-6) exhibited severe malformation of the head. Embryos exposed later (HH 6-9) manifested spina bifida at the thoracic and lumbosacral levels. Mirror image heart looping was also observed in 20% of these embryos. Paraxial mesoderm was apparently unaffected. Changes in cell proliferation and induced cell death preceded morphological changes. We used acridine orange and confocal laser microscopy to demonstrate that hyperthermia induced cell death in neural folds starting 6 h following treatment. To assess cell proliferation, we used BrdU incorporation for 4 h. Immunodetection on paraffin sections demonstrated that proliferation was inhibited 6 h after treatment. Heat-exposed embryos exhibited the heat shock response, with protein expression reaching a maximum 4-6 h following heat treatment. Malformed embryos showed an intense heat shock response for a further 6 h. The levels of induced heat shock proteins were similar in the affected neural tube and in the heart, where neither induced cell death nor malformations were observed.

Spatial arrangement of fetal placental capillaries in terminal villi: a study using confocal microscopy.

The spatial arrangement of capillaries was studied in terminal villi of placentas at term by using confocal microscopy and methods for different types of 3-D reconstructions. Fixed specimens embedded in paraffin or glycol methacrylate resin were cut and stained with eosin. Digitized images of serial optical sections (approximately 0.5 microm) of individual terminal villi lying completely inside physical sections (100 microm) were captured by confocal laser scanning microscopy and analyzed. Topological reconstruction of the capillary bed and measurements of its Euler number, surface and volume rendering and wire-frame visualization were performed. Our findings showed that villous capillaries are arranged either in a single loop or in a more or less complicated anastomosing system. The results suggest that the combination of confocal microscope capture, methods for 3-D rendering and characterization of topological features reveals valuable information about spatial relationships of tissues in placental villi and the arrangement of the villous microcirculation, e.g. about the branching pattern of capillaries.