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Showing their true colors: a practical approach to volume rendering from serial sections.

ABSTRACT: BACKGROUND: In comparison to more modern imaging methods, conventional light microscopy still offers a range of substantial advantages with regard to contrast options, accessible specimen size, and resolution. Currently, tomographic image data in particular is most commonly visualized in three dimensions using volume rendering. To date, this method has only very rarely been applied to image stacks taken from serial sections, whereas surface rendering is still the most prevalent method for presenting such data sets three-dimensionally. The aim of this study was to develop standard protocols for volume rendering of image stacks of serial sections, while retaining the benefits of light microscopy such as resolution and color information. RESULTS: Here we provide a set of protocols for acquiring high-resolution 3D images of diverse microscopic samples through volume rendering based on serial light microscopical sections using the 3D reconstruction software Amira (Visage Imaging Inc.). We overcome several technical obstacles and show that these renderings are comparable in quality and resolution to 3D visualizations using other methods. This practical approach for visualizing 3D micro-morphology in full color takes advantage of both the sub-micron resolution of light microscopy and the specificity of histological stains, by combining conventional histological sectioning techniques, digital image acquisition, three-dimensional image filtering, and 3D image manipulation and visualization technologies. CONCLUSIONS: We show that this method can yield "true"-colored high-resolution 3D views of tissues as well as cellular and sub-cellular structures and thus represents a powerful tool for morphological, developmental, and comparative investigations. We conclude that the presented approach fills an important gap in the field of micro-anatomical 3D imaging and visualization methods by combining histological resolution and differentiation of details with 3D rendering of whole tissue samples. We demonstrate the method on selected invertebrate and vertebrate specimens, and propose that reinvestigation of historical serial section material may be regarded as a special benefit.

SUBMITTER: Handschuh S

PROVIDER: S-EPMC2876090 | biostudies-other | 2010

REPOSITORIES: biostudies-other

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Showing their true colors: a practical approach to volume rendering from serial sections.

Handschuh Stephan S Schwaha Thomas T Metscher Brian D BD

BMC developmental biology 20100421

<h4>Background</h4>In comparison to more modern imaging methods, conventional light microscopy still offers a range of substantial advantages with regard to contrast options, accessible specimen size, and resolution. Currently, tomographic image data in particular is most commonly visualized in three dimensions using volume rendering. To date, this method has only very rarely been applied to image stacks taken from serial sections, whereas surface rendering is still the most prevalent method for ...[more]

PMID: 20409315

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Project description:Ticks are important ectoparasites that are capable of transmitting multiple classes of pathogens and are currently linked with many emerging tick-borne diseases worldwide. With increasing occurrences of tick-borne diseases in both humans and veterinary species, there is a continuous need to further our understanding of ticks and the pathogens they transmit. Whole tick histology provides a full scope of the tick internal anatomy, allowing researchers to examine multiple organs of interest in a single section. This is in contrast to other techniques that are more commonly utilized in tick-borne disease research, such as electron microscopy and light microscopy of individual organs. There is a lack of literature describing a practical technique to process whole tick histologic sections. Therefore, the current study aims to provide researchers with a workable protocol to prepare high quality paraffin-embedded whole tick histology sections. Amblyomma americanum adults were used as an example species for this study. After a series of pilot experiments using a combination of various fixatives, softening agents and processing techniques, we elected to compare two common fixatives, 10% neutral-buffered formalin (NBF) and Bouin's solution for whole ticks. Equal numbers of A. americanum unfed adults (n = 10/fixative) were processed identically and their whole tick histology coronal sections were individually scored. Higher scores were assigned to whole tick sections that contained more internal organs that are crucial for tick-borne disease research (e.g. salivary glands and midgut), high integrity of tissues and exoskeleton on the section, and good fixation and staining quality of the tissues. The mean total scores for Bouin's-fixed ticks were significantly higher compared to NBF-fixed ticks (p = 0.001). To further assess our preferred technique, we also demonstrated the feasibility of producing high quality whole tick sections for three other common tick species of medical importance (Rhipicephalus sanguineus, Ixodes scapularis, and Dermacentor variabilis) using Bouin's solution. While this technique may require further optimization for other tick species, we described a feasible protocol that uses commonly available tools, reagents and standard histologic equipment. This should allow any investigator to easily make adjustments to this protocol as needed based on their experimental goals.

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Showing their true colors: a practical approach to volume rendering from serial sections.

Publications

Showing their true colors: a practical approach to volume rendering from serial sections.

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