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Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine.

Lee H, Park JH, Seo I, Park SH, Kim S - BMC Dev. Biol. (2014)

Bottom Line: We determined the optimal conditions for reducing bubble formation, discoloration, and depositing of black particles on the surface of tissue, which allowed production of clearer organ images.We developed improved CLARITY methods for clearing of the brain, pancreas, lung, intestine, liver, and kidney, and identified the appropriate experimental conditions for clearing of each specific tissue type.These optimized methods will be useful for the application of CLARITY to various types of organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Keimyung University School of Medicine, 1095 Dalgubeoldae-Ro, Dalseo-Gu, Daegu, 704-701, South Korea. neuroana@dsmc.or.kr.

ABSTRACT

Background: Mapping of tissue structure at the cellular, circuit, and organ-wide scale is important for understanding physiological and biological functions. A bio-electrochemical technique known as CLARITY used for three-dimensional anatomical and phenotypical mapping within transparent intact tissues has been recently developed. This method provided a major advance in understanding the structure-function relationships in circuits of the nervous system and organs by using whole-body clearing. Thus, in the present study, we aimed to improve the original CLARITY procedure and developed specific CLARITY protocols for various intact organs.

Results: We determined the optimal conditions for reducing bubble formation, discoloration, and depositing of black particles on the surface of tissue, which allowed production of clearer organ images. We also determined the appropriate replacement cycles of clearing solution for each type of organ, and convincingly demonstrated that 250-280 mA is the ideal range of electrical current for tissue clearing. We then acquired each type of cleared organs including brain, pancreas, liver, lung, kidney, and intestine. Additionally, we determined the images of axon fibers of hippocampal region, the Purkinje layer of cerebellum, and vessels and cellular nuclei of pancreas.

Conclusions: CLARITY is an innovative biochemical technology for the structural and molecular analysis of various types of tissue. We developed improved CLARITY methods for clearing of the brain, pancreas, lung, intestine, liver, and kidney, and identified the appropriate experimental conditions for clearing of each specific tissue type. These optimized methods will be useful for the application of CLARITY to various types of organs.

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Pancreatic vessel imaging in the intact adult mouse pancreas. In adult mouse tissues (12 weeks old), imaging was performed after CLARITY. (A) Three-dimensional (3D) projection (left panel) and rendering (right panel) of clarified mouse pancreas without capillary immunostained for α-smooth muscle actin (green). Scale bar, 300 μm (Additional files 3 and 4). (B) Three-dimensional projection (left panel) clarified mouse pancreas with capillary immunostained for α-smooth muscle actin (green). Scale bar, 200 μm. Merged image with manually traced (green, middle panel) and 3D rendering extracted capillary (right panel). Representative data were chosen from five independent experiments.
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Fig3: Pancreatic vessel imaging in the intact adult mouse pancreas. In adult mouse tissues (12 weeks old), imaging was performed after CLARITY. (A) Three-dimensional (3D) projection (left panel) and rendering (right panel) of clarified mouse pancreas without capillary immunostained for α-smooth muscle actin (green). Scale bar, 300 μm (Additional files 3 and 4). (B) Three-dimensional projection (left panel) clarified mouse pancreas with capillary immunostained for α-smooth muscle actin (green). Scale bar, 200 μm. Merged image with manually traced (green, middle panel) and 3D rendering extracted capillary (right panel). Representative data were chosen from five independent experiments.

Mentions: We performed immunostaining in adult mouse brain using a microtubule-associated protein tau-antibody. Using confocal images, tau-stained axon fiber was confirmed in the hippocampal region (Additional file 2) and the Purkinje layer of cerebellum (Figure 2). As shown in Additional file 2, the hippocampal region was reconstructed in 3D stacks of images (Z-stack volume, 650 μm). Moreover, the Purkinje layer of cerebellum was reconstructed in 3D stacks of images (Z-stack volume, 210 μm) (Additional file 3 and Additional file 4). Furthermore, we performed imaging in pancreas sample with an α-smooth muscle actin-antibody which is the marker for blood vessels including capillary vessels [8]. We could identify stained vessels in randomly selected regions of distal pancreas in optically transparent pancreas (Figure 3A, Additional files 5, 6, 7, 8 and 9). The vessel and pancreatic region was reconstructed in 3D stacks of images (Z-stack volume, 650 μm). To reconstruct the 3D morphology of the pancreatic capillaries, we traced the profile of the α-smooth muscle actin and extracted the image of the pancreatic capillary using ImageJ (Figure 3B, Additional file 10). To identify the structural integrity of cells-vasculature relationships in distal region of the pancreas, we fluorescent stained the cleared pancreas samples with an α-smooth muscle actin-antibody and DAPI, which binds strongly to A-T rich regions in DNA. We could identify stained vessels and pancreatic cellular nuclei in randomly selected regions of the distal pancreas in optically transparent pancreas (Z-stack volume, 110 μm) (Figure 4, Additional file 11).Figure 2


Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine.

Lee H, Park JH, Seo I, Park SH, Kim S - BMC Dev. Biol. (2014)

Pancreatic vessel imaging in the intact adult mouse pancreas. In adult mouse tissues (12 weeks old), imaging was performed after CLARITY. (A) Three-dimensional (3D) projection (left panel) and rendering (right panel) of clarified mouse pancreas without capillary immunostained for α-smooth muscle actin (green). Scale bar, 300 μm (Additional files 3 and 4). (B) Three-dimensional projection (left panel) clarified mouse pancreas with capillary immunostained for α-smooth muscle actin (green). Scale bar, 200 μm. Merged image with manually traced (green, middle panel) and 3D rendering extracted capillary (right panel). Representative data were chosen from five independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4281481&req=5

Fig3: Pancreatic vessel imaging in the intact adult mouse pancreas. In adult mouse tissues (12 weeks old), imaging was performed after CLARITY. (A) Three-dimensional (3D) projection (left panel) and rendering (right panel) of clarified mouse pancreas without capillary immunostained for α-smooth muscle actin (green). Scale bar, 300 μm (Additional files 3 and 4). (B) Three-dimensional projection (left panel) clarified mouse pancreas with capillary immunostained for α-smooth muscle actin (green). Scale bar, 200 μm. Merged image with manually traced (green, middle panel) and 3D rendering extracted capillary (right panel). Representative data were chosen from five independent experiments.
Mentions: We performed immunostaining in adult mouse brain using a microtubule-associated protein tau-antibody. Using confocal images, tau-stained axon fiber was confirmed in the hippocampal region (Additional file 2) and the Purkinje layer of cerebellum (Figure 2). As shown in Additional file 2, the hippocampal region was reconstructed in 3D stacks of images (Z-stack volume, 650 μm). Moreover, the Purkinje layer of cerebellum was reconstructed in 3D stacks of images (Z-stack volume, 210 μm) (Additional file 3 and Additional file 4). Furthermore, we performed imaging in pancreas sample with an α-smooth muscle actin-antibody which is the marker for blood vessels including capillary vessels [8]. We could identify stained vessels in randomly selected regions of distal pancreas in optically transparent pancreas (Figure 3A, Additional files 5, 6, 7, 8 and 9). The vessel and pancreatic region was reconstructed in 3D stacks of images (Z-stack volume, 650 μm). To reconstruct the 3D morphology of the pancreatic capillaries, we traced the profile of the α-smooth muscle actin and extracted the image of the pancreatic capillary using ImageJ (Figure 3B, Additional file 10). To identify the structural integrity of cells-vasculature relationships in distal region of the pancreas, we fluorescent stained the cleared pancreas samples with an α-smooth muscle actin-antibody and DAPI, which binds strongly to A-T rich regions in DNA. We could identify stained vessels and pancreatic cellular nuclei in randomly selected regions of the distal pancreas in optically transparent pancreas (Z-stack volume, 110 μm) (Figure 4, Additional file 11).Figure 2

Bottom Line: We determined the optimal conditions for reducing bubble formation, discoloration, and depositing of black particles on the surface of tissue, which allowed production of clearer organ images.We developed improved CLARITY methods for clearing of the brain, pancreas, lung, intestine, liver, and kidney, and identified the appropriate experimental conditions for clearing of each specific tissue type.These optimized methods will be useful for the application of CLARITY to various types of organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Keimyung University School of Medicine, 1095 Dalgubeoldae-Ro, Dalseo-Gu, Daegu, 704-701, South Korea. neuroana@dsmc.or.kr.

ABSTRACT

Background: Mapping of tissue structure at the cellular, circuit, and organ-wide scale is important for understanding physiological and biological functions. A bio-electrochemical technique known as CLARITY used for three-dimensional anatomical and phenotypical mapping within transparent intact tissues has been recently developed. This method provided a major advance in understanding the structure-function relationships in circuits of the nervous system and organs by using whole-body clearing. Thus, in the present study, we aimed to improve the original CLARITY procedure and developed specific CLARITY protocols for various intact organs.

Results: We determined the optimal conditions for reducing bubble formation, discoloration, and depositing of black particles on the surface of tissue, which allowed production of clearer organ images. We also determined the appropriate replacement cycles of clearing solution for each type of organ, and convincingly demonstrated that 250-280 mA is the ideal range of electrical current for tissue clearing. We then acquired each type of cleared organs including brain, pancreas, liver, lung, kidney, and intestine. Additionally, we determined the images of axon fibers of hippocampal region, the Purkinje layer of cerebellum, and vessels and cellular nuclei of pancreas.

Conclusions: CLARITY is an innovative biochemical technology for the structural and molecular analysis of various types of tissue. We developed improved CLARITY methods for clearing of the brain, pancreas, lung, intestine, liver, and kidney, and identified the appropriate experimental conditions for clearing of each specific tissue type. These optimized methods will be useful for the application of CLARITY to various types of organs.

Show MeSH