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Title: 3D reconstructions with pixel-based images are made possible by digitally clearing plant and animal tissue

item Livingston, David
item Tuong, Tan
item GADI, SASHI - North Carolina State University
item HAIGLER, CANDACE - North Carolina State University
item CULLEN, JOHN - North Carolina State University
item GELMAN, RANDY - Beltline Media

Submitted to: Journal of Microscopy
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2010
Publication Date: 10/1/2010
Citation: Livingston, D.P., Tuong, T.D., Gadi, S., Haigler, C., Cullen, J., Gelman, R. 2010. 3D reconstructions with pixel-based images are made possible by digitally clearing plant and animal tissue. Journal of Microscopy. 240:122-129.

Interpretive Summary: The scientific and medical community has been using techniques such as Magnetic resonance imaging (MRI), positron emission tomography/computed tomography (PET/CT) Syncrotron Radiation Imaging (SRI) and confocal laser scanning microscopy (CLSC) as well as a software technique called “contour tracing” to reconstruct internal structures in biological tissue in 3 dimensions. However, these technologies are based on the ability to generate 2 dimensional images with vector-based information. Each of these technologies has short-comings that will not allow them to be used with small structures such as plants or biopsies of human or animal tissue. Most histological images generated in laboratories researching smaller tissues rely on pixel-based images which until now could not be used to generate 3D images. We have developed a workflow using Adobe After Effects that for the first time will allow 3D reconstruction from any series of images generated with or without a microscope. While it will not replace the existing technologies described above, it makes possible the viewing of internal structures from images generated from inexpensive digital consumer-grade photographic equipment. This new 3D reconstruction method is based on a common visual effects technique known as “chroma-keying” in which any color in an image can be removed allowing the image to become transparent. When a series of images are then aligned and rotated in 3D space, virtually any object of interest within the tissue may be observed in 3 dimensions. We demonstrate its versatility by showing a necrotic tumor in the liver of a woodchuck, internal damage caused by freezing within an oat crown, and various internal structures within a small piece of mouse lung.

Technical Abstract: Reconstruction of 3D images from a series of 2D images has been restricted by the limited capacity to decrease the opacity of surrounding tissue. Commercial software that allows color-keying and manipulation of 2D images in true 3D space allowed us to produce 3D reconstructions from pixel based images of stained plant and animal tissue without the need to generate vector information. We present 3D reconstructions of: 1) a sample of a hepatocellular carcinoma from a woodchuck liver that had been heat-treated with computer-guided radiofrequency ablation to induce necrosis in the central portion of the tumor: 2) the crown of an oat plant showing internal damage caused by freezing: and 3) several features of a sample of mouse lung. The technique described is widely applicable, having potential to generate 3D images at virtually any resolution inherent in serial images generated by light microscopy, computer tomography, magnetic resonance imaging or electron microscopy.