Cellular maps of gastrointestinal organs: getting the most from tissue clearing.

The development of modern methods to induce optical transparency ("clearing") in biological tissues has enabled the three-dimensional (3D) reconstruction of intact organs at cellular resolution. New capabilities in visualization of rare cellular events, long-range interactions, and irregular structures will facilitate novel studies in the alimentary tract and gastrointestinal systems. The tubular geometry of the alimentary tract facilitates large-scale cellular reconstruction of cleared tissue without specialized microscopy setups. However, with the rapid pace of development of clearing agents and current relative paucity of research groups in the gastrointestinal field using these techniques, it can be daunting to incorporate tissue clearing into experimental workflows. Here, we give some advice and describe our own experience bringing tissue clearing and whole mount reconstruction into our laboratory's investigations. We present a brief overview of the chemical concepts that underpin tissue clearing, what sorts of questions whole mount imaging can answer, how to choose a clearing agent, an example of how to clear and image alimentary tissue, and what to do after obtaining the image. This short review will encourage other gastrointestinal researchers to consider how utilizing tissue clearing and creating 3D "maps" of tissue might deepen the impact of their studies.

Directional Histogram Ratio at Random Probes: A Local Thresholding Criterion for Capillary Images.

With the development of micron-scale imaging techniques, capillaries can be conveniently visualized using methods such as two-photon and whole mount microscopy. However, the presence of background staining, leaky vessels and the diffusion of small fluorescent molecules can lead to significant complexity in image analysis and loss of information necessary to accurately quantify vascular metrics. One solution to this problem is the development of accurate thresholding algorithms that reliably distinguish blood vessels from surrounding tissue. Although various thresholding algorithms have been proposed, our results suggest that without appropriate pre- or post-processing, the existing approaches may fail to obtain satisfactory results for capillary images that include areas of contamination. In this study, we propose a novel local thresholding algorithm, called directional histogram ratio at random probes (DHR-RP). This method explicitly considers the geometric features of tube-like objects in conducting image binarization, and has a reliable performance in distinguishing small vessels from either clean or contaminated background. Experimental and simulation studies suggest that our DHR-RP algorithm is superior over existing thresholding methods.