Stereology as the 3D tool to quantitate lung architecture

Stereology is the method of choice for the quantitative assessment of biological objects in microscopy. It takes into account the fact that, in traditional microscopy such as conventional light and transmission electron microscopy, although one has to rely on measurements on nearly two-dimensional sections from fixed and embedded tissue samples, the quantitative data obtained by these measurements should characterize the real three-dimensional properties of the biological objects and not just their "flatland" appearance on the sections. Thus, three-dimensionality is a built-in property of stereological sampling and measurement tools. Stereology is, therefore, perfectly suited to be combined with 3D imaging techniques which cover a wide range of complementary sample sizes and resolutions, e.g. micro-computed tomography, confocal microscopy and volume electron microscopy. Here, we review those stereological principles that are of particular relevance for 3D imaging and provide an overview of applications of 3D imaging-based stereology to the lung in health and disease. The symbiosis of stereology and 3D imaging thus provides the unique opportunity for unbiased and comprehensive quantitative characterization of the three-dimensional architecture of the lung from macro to nano scale

THREE-DIMENSIONAL RECONSTRUCTION AND VISUALIZATION OF PLANT CELLS

The mechanical properties (like sensory texture etc.) of plants/fruits directly depend on their microstructures. Therefore, it is very important to well understand the geometry and topology of cells in order to control the microstructure for better mechanical response. In this research, techniques of digital image processing and segmentation in conjunction with mathematical morphology models are used to visualize and analyze the 3D cells of potato. ImageJ and MATLAB are used throughout in this study. The labeled image stacks are essential for studying quantitative characterization of 3D cells, MATLAB is used to label each image stacks. By using MATLAB 12420 cells were segmented within a short period of time and labeled each cell uniquely

Confocal Microscopy and Three-Dimensional Reconstruction of Thick, Transparent, Vital Tissue

The three-dimensional visualization of the 400 micron thick, transparent, in situ cornea is described to demonstrate the use of confocal light microscopy for noninvasive imaging of living cells and thick tissues in their normal, vital conditions. Specimen preparation and physiological stability, as well as light attenuation corrections are critical to data acquisition. The technique to provide mechanical stability of the specimen during the duration of the image acquisition is explained. A laser scanning confocal light microscope (LSCM) was used to obtain optical serial sections from rabbit eyes that were freshly removed and placed in a physiological Ringer\u27s solution. This study demonstrates the capability of the confocal light microscope to obtain a series of high contrast images, with a depth resolution of one micron, across the full thickness of living, transparent tissue. The problems of nonisotropic sampling and the limited eight-bit dynamic range are discussed. The three-dimensional reconstructions were obtained by computer graphics using the volume visualization projection technique. The three-dimensional visualization of the cornea in the in situ eye is presented as an example of image understanding of thick, viable biological cells and tissues. Finally, the criterion of image fidelity is explained. The techniques of confocal light microscopy with its enhanced lateral and axial resolution, improved image contrast, and volume visualization provides microscopists with new techniques for the observation of vital cells and tissues, both in vivo and in vitro

Histopathological Morphometry of Human Endobronchial Biopsies – a Comparison of Conventional Quantitative Analyses and Stereological Designs

Endobronchial biopsies collected by fiberoptic bronchoscopy have been increasingly used in biomedical research on disease mechanisms and clinical therapy studies of chronic inflammatory airway disorders. Although less invasive techniques are available for the investigation of the inflammatory infiltrate of the bronchial tree, a standardization of their results with respect to the extent or level of the sampled airway proved impracticable. Moreover in a clinical setting the structural alterations of the airway mucosa can only be assessed by histopathological biopsy analysis, which makes this approach indispensable to airway research. More and more quantitative approaches in biopsy studies have been reported. The high variability of their results points out the need for reliable and robust quantitative methods and sampling designs in order to allow for an easier interpretation and corroboration of the outcomes of different studies. It is unclear whether classical 2D approaches and unbiased stereological 3D designs for counting inflammatory cells, measuring area fraction or layer thickness on histological sections are equally well suited for these purposes. The aim of this study was to characterise the agreement between 2D and 3D approaches for inflammatory cell counting by simultaneously applying them on bioptic material. Furthermore, stereological designs were proposed for quantifying the extent of epithelial desquamation and the mean thickness of the reticular basement membrane, and the results were related to previously published data gained by 2D tissue analyses. The hypotheses that the epithelial integrity depends on biopsy size or mean basement membrane thickness were also verified. Biopsies from the segmental bronchi were collected by fiberoptic bronchoscopy in a group of smokers (n=7) and a group of healthy non-smokers (n=7), embedded in paraffin and exhaustively sectioned. Systematic uniform random samples of sections were stained histochemically (PAS) or immunohistochemically for macrophages (CD68) and T-lymphocytes (CD3), respectively. On the same systematic uniform random samples of fields of view, cell numbers per unit volume were assessed using the physical disector and cell and nuclear profiles were counted and related to the subepithelial layer area. To obtain a zero-dimensional index allowing for a direct comparison of the two methods, the CD68+/CD3+ ratio was calculated for each approach. The extent of epithelial desquamation was assessed as area fraction of the basement membrane by counting the intersections of a line grid with the basement membrane on PAS stained sections. On the same sections the arithmetic mean thickness of the reticular basement membrane was estimated using a coherent test system of points and line segments. Counting cell profiles per unit area severely overestimated the number of larger cells (macrophages) relative to smaller cells (T-lymphocytes). Counting of nuclear profiles delivered average values similar to the physical disector but a bias proportional to the magnitude of the CD68+/CD3+ ratios was identified. The extent of epithelial desquamation was similar between the two groups and in accordance with previous studies in healthy volunteers and asthmatics. The lack of a difference between the (non-asthmatic) subjects of this study and published data on asthma patients confirms earlier similar findings. This strengthens the doubt about the morphopathological significance of the epithelial disruption, suggesting an artefactual cause. The arithmetic mean thickness of the reticular basement membrane, an important marker of airway remodelling in biopsy studies of asthma, showed no significant difference between healthy non-smokers and smokers in the small studied groups. The average values were very similar to the results of another published stereological design and to those obtained by image analysis of perpendicular sections. At the same time they were conspicuously lower than the data reported by studies employing direct point-to-point measurements on sections. This underlines the overestimation of the mean thickness introduced by tangential cutting of the basement membrane when relying on 2D measurements of this three-dimensional structure

Benchmarking of tools for axon length measurement in individually-labeled projection neurons

Projection neurons are the commonest neuronal type in the mammalian forebrain and their individual characterization is a crucial step to understand how neural circuitry operates. These cells have an axon whose arborizations extend over long distances, branching in complex patterns and/or in multiple brain regions. Axon length is a principal estimate of the functional impact of the neuron, as it directly correlates with the number of synapses formed by the axon in its target regions; however, its measurement by direct 3D axonal tracing is a slow and labor-intensive method. On the contrary, axon length estimations have been recently proposed as an effective and accessible alternative, allowing a fast approach to the functional significance of the single neuron. Here, we analyze the accuracy and efficiency of the most used length estimation tools—design-based stereology by virtual planes or spheres, and mathematical correction of the 2D projected-axon length—in contrast with direct measurement, to quantify individual axon length. To this end, we computationally simulated each tool, applied them over a dataset of 951 3D-reconstructed axons (from NeuroMorpho.org), and compared the generated length values with their 3D reconstruction counterparts. The evaluated reliability of each axon length estimation method was then balanced with the required human effort, experience and know-how, and economic affordability. Subsequently, computational results were contrasted with measurements performed on actual brain tissue sections. We show that the plane-based stereological method balances acceptable errors (~5%) with robustness to biases, whereas the projection-based method, despite its accuracy, is prone to inherent biases when implemented in the laboratory. This work, therefore, aims to provide a constructive benchmark to help guide the selection of the most efficient method for measuring specific axonal morphologies according to the particular circumstances of the conducted research

Automated Correlative Light and Electron Microscopy using FIB-SEM as a tool to screen for ultrastructural phenotypes

In Correlative Light and Electron Microscopy (CLEM), two imaging modalities are combined to take advantage of the localization capabilities of light microscopy (LM) to guide the capture of high-resolution details in the electron microscope (EM). However, traditional approaches have proven to be very laborious, thus yielding a too low throughput for quantitative or exploratory studies of populations. Recently, in the electron microscopy field, FIB-SEM (Focused Ion Beam -Scanning Electron Microscope) tomography has emerged as a flexible method that enables semi-automated 3D volume acquisitions. During my thesis, I developed CLEMSite, a tool that takes advantage of the semi-automation and scanning capabilities of the FIB-SEM to automatically acquire volumes of adherent cultured cells. CLEMSite is a combination of computer vision and machine learning applications with a library for controlling the microscope ( product from a collaboration with Carl Zeiss GmbH and Fibics Inc.). Thanks to this, the microscope was able to automatically track, find and acquire cell regions previously identified in the light microscope. More specifically, two main modules were implemented. First, a correlation module was designed to detect and record reference points from a grid pattern present on the culture substrate in both modalities (LM and EM). Second, I designed a module that retrieves the regions of interest in the FIB-SEM and that drives the acquisition of image stacks between different targets in an unattended fashion. The automated CLEM approach is demonstrated on a project where 3D EM volumes are examined upon multiple siRNA treatments for knocking down genes involved in the morphogenesis of the Golgi apparatus. Additionally, the power of CLEM approaches using FIB-SEM is demonstrated with the detailed structural analysis of two events: the breakage of the nuclear envelope within constricted cells and an intriguing catastrophic DNA Damage Response in binucleated cells. Our results demonstrate that executing high throughput volume acquisition in electron microscopy is possible and that EM can provide incredible insights to guide new biological discoveries