Abstract
We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining.
Highlights
Whole Slide Imaging (WSI) systems are commercially available from various companies and may be used to collect high-resolution widefield microscope images, current virtual slide systems are mostly limited to brightfield and fluorescence imaging; they do not provide high magnification darkfield imaging
Imaging of DAB-stained Specimens Using darkfield internal reflection illumination (DIRI) DAB-stained brain sections were imaged with a 206 objective, using brightfield mode (Figure 5A), darkfield mode using DIRI (Figure 5B) and a traditional darkfield substage condenser, which was aligned and focused to conform to principles of Koehler illumination (Figure 5C)
To compare the DIRI images, with traditional darkfield, we examined the same specimens with dust on the top of the cover slip, using a darkfield substage condenser with a 206 objective lens (Figure 5E)
Summary
In order to compensate for the optical and emulsion deficiencies of their photographic apparatus, they attempted to capture the complexity of the neural tissue by using a microscope drawing tube to sketch a high-resolution image in widefield [1]. One of the most powerful applications of WSI is the ability to scan a complete set of serial sections within a brief period of time This advantage greatly simplifies the task of analysis and allows researchers to share the research results with colleagues thousands of miles away. WSI systems are commercially available from various companies and may be used to collect high-resolution widefield microscope images, current virtual slide systems are mostly limited to brightfield and fluorescence imaging; they do not provide high magnification darkfield imaging. We sought to develop a method of combining darkfield imaging with WSI technology to overcome these limitations
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