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Procedures for the quantification of whole-tissue immunofluorescence images obtained at single-cell resolution during murine tubular organ development.

Hirashima T, Adachi T - PLoS ONE (2015)

Bottom Line: However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed.Through comparison of fixative solutions and of clearing methods, we found optimal conditions to achieve clearer deep-tissue imaging of specific immunolabeled targets and explain what methods result in vivid volume imaging.The procedure for the whole-tissue quantification shown in this article should contribute to systematic measurements of cellular processes in developing organs, accelerating the further understanding of morphogenesis at the single cell level.

View Article: PubMed Central - PubMed

Affiliation: Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

ABSTRACT
Whole-tissue quantification at single-cell resolution has become an inevitable approach for further quantitative understanding of morphogenesis in organ development. The feasibility of the approach has been dramatically increased by recent technological improvements in optical tissue clearing and microscopy. However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed. To provide the appropriate procedure, we here show tips for each critical step of the entire process, including fixation for immunofluorescence, optical clearing, and digital image processing, using developing murine internal organs such as epididymis, kidney, and lung as an example. Through comparison of fixative solutions and of clearing methods, we found optimal conditions to achieve clearer deep-tissue imaging of specific immunolabeled targets and explain what methods result in vivid volume imaging. In addition, we demonstrated that three-dimensional digital image processing after optical clearing produces objective quantitative data for the whole-tissue analysis, focusing on the spatial distribution of mitotic cells in the epididymal tubule. The procedure for the whole-tissue quantification shown in this article should contribute to systematic measurements of cellular processes in developing organs, accelerating the further understanding of morphogenesis at the single cell level.

No MeSH data available.


Related in: MedlinePlus

Comparison of immunofluorescence for E-cadherin among optical clearing methods.Different optical clearing methods, including BABB, SeeDB, CUBIC, and PACT, were applied to the embryonic epididymis (E18.5), kidney (E16.5), and lung (E15.5), each of which was immunolabeled with E-cadherin. PBS was used as a control solution. The color represents the optical clearing method. (A) Plot of the mean E-cadherin immunofluorescence intensity against the depth from the bottom of tissues embedded onto the dish. The mean intensity was normalized to the maximum intensity of PBS-treated samples (black) in each organ. n = 8. The error bars represent the standard deviation (s.d.). (B) Representative images of E-cadherin immunofluorescence in the epididymis are shown for each depth from the bottom of the tissue (row) and for each optical clearing method (column). Scale bar: 50 μm. (C) Plot of the mean E-cadherin immunofluorescence intensity against the depth from the bottom of tissues between CUBIC and CUBIC-BABB. n = 8. The error bars represent s.d. (D) Mean value of maximum intensity in each tissue between CUBIC and CUBIC-BABB. n = 8. P value > 0.05 for epididymis, kidney and lung (Welch’s t test). P values of less than 0.05 were considered to be statistically significant. The error bars represent s.d.
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pone.0135343.g003: Comparison of immunofluorescence for E-cadherin among optical clearing methods.Different optical clearing methods, including BABB, SeeDB, CUBIC, and PACT, were applied to the embryonic epididymis (E18.5), kidney (E16.5), and lung (E15.5), each of which was immunolabeled with E-cadherin. PBS was used as a control solution. The color represents the optical clearing method. (A) Plot of the mean E-cadherin immunofluorescence intensity against the depth from the bottom of tissues embedded onto the dish. The mean intensity was normalized to the maximum intensity of PBS-treated samples (black) in each organ. n = 8. The error bars represent the standard deviation (s.d.). (B) Representative images of E-cadherin immunofluorescence in the epididymis are shown for each depth from the bottom of the tissue (row) and for each optical clearing method (column). Scale bar: 50 μm. (C) Plot of the mean E-cadherin immunofluorescence intensity against the depth from the bottom of tissues between CUBIC and CUBIC-BABB. n = 8. The error bars represent s.d. (D) Mean value of maximum intensity in each tissue between CUBIC and CUBIC-BABB. n = 8. P value > 0.05 for epididymis, kidney and lung (Welch’s t test). P values of less than 0.05 were considered to be statistically significant. The error bars represent s.d.

Mentions: The contents of this article are organized according to the sequence of the standard protocol for the whole-tissue quantification and cover only the critical steps described in Table 1. Throughout this article, we focus on developing murine tubular organs, such as epididymis, kidney, and lung. Although these organs are different in terms of shape and function, they have a common structure that a single-layered epithelial tubule is embedded in mesenchyme and extra-cellular matrix, enveloped by a clear border of organ. In the first section of the Results, we show typical images obtained through whole-tissue fluorescence immunolabeling for F-actin and E-cadherin in epithelial tubules. In the next section, we present a case, in which the fixative solutions significantly alter the labeling performance on pMLC immunofluorescence. This section is aimed to advise the readers to pay general attention to the choice of fixative in the process of whole-tissue quantification. Then, we compare established optical clearing methods regarding how much signal intensity can be detected in the deep region of tissues, and examine whether the combination of the established clearing methods can improve deep tissue imaging. Finally, automatic whole-tissue quantification for mitotic cells by digital image processing is demonstrated using the embryonic murine epididymal tubule.


Procedures for the quantification of whole-tissue immunofluorescence images obtained at single-cell resolution during murine tubular organ development.

Hirashima T, Adachi T - PLoS ONE (2015)

Comparison of immunofluorescence for E-cadherin among optical clearing methods.Different optical clearing methods, including BABB, SeeDB, CUBIC, and PACT, were applied to the embryonic epididymis (E18.5), kidney (E16.5), and lung (E15.5), each of which was immunolabeled with E-cadherin. PBS was used as a control solution. The color represents the optical clearing method. (A) Plot of the mean E-cadherin immunofluorescence intensity against the depth from the bottom of tissues embedded onto the dish. The mean intensity was normalized to the maximum intensity of PBS-treated samples (black) in each organ. n = 8. The error bars represent the standard deviation (s.d.). (B) Representative images of E-cadherin immunofluorescence in the epididymis are shown for each depth from the bottom of the tissue (row) and for each optical clearing method (column). Scale bar: 50 μm. (C) Plot of the mean E-cadherin immunofluorescence intensity against the depth from the bottom of tissues between CUBIC and CUBIC-BABB. n = 8. The error bars represent s.d. (D) Mean value of maximum intensity in each tissue between CUBIC and CUBIC-BABB. n = 8. P value > 0.05 for epididymis, kidney and lung (Welch’s t test). P values of less than 0.05 were considered to be statistically significant. The error bars represent s.d.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4530862&req=5

pone.0135343.g003: Comparison of immunofluorescence for E-cadherin among optical clearing methods.Different optical clearing methods, including BABB, SeeDB, CUBIC, and PACT, were applied to the embryonic epididymis (E18.5), kidney (E16.5), and lung (E15.5), each of which was immunolabeled with E-cadherin. PBS was used as a control solution. The color represents the optical clearing method. (A) Plot of the mean E-cadherin immunofluorescence intensity against the depth from the bottom of tissues embedded onto the dish. The mean intensity was normalized to the maximum intensity of PBS-treated samples (black) in each organ. n = 8. The error bars represent the standard deviation (s.d.). (B) Representative images of E-cadherin immunofluorescence in the epididymis are shown for each depth from the bottom of the tissue (row) and for each optical clearing method (column). Scale bar: 50 μm. (C) Plot of the mean E-cadherin immunofluorescence intensity against the depth from the bottom of tissues between CUBIC and CUBIC-BABB. n = 8. The error bars represent s.d. (D) Mean value of maximum intensity in each tissue between CUBIC and CUBIC-BABB. n = 8. P value > 0.05 for epididymis, kidney and lung (Welch’s t test). P values of less than 0.05 were considered to be statistically significant. The error bars represent s.d.
Mentions: The contents of this article are organized according to the sequence of the standard protocol for the whole-tissue quantification and cover only the critical steps described in Table 1. Throughout this article, we focus on developing murine tubular organs, such as epididymis, kidney, and lung. Although these organs are different in terms of shape and function, they have a common structure that a single-layered epithelial tubule is embedded in mesenchyme and extra-cellular matrix, enveloped by a clear border of organ. In the first section of the Results, we show typical images obtained through whole-tissue fluorescence immunolabeling for F-actin and E-cadherin in epithelial tubules. In the next section, we present a case, in which the fixative solutions significantly alter the labeling performance on pMLC immunofluorescence. This section is aimed to advise the readers to pay general attention to the choice of fixative in the process of whole-tissue quantification. Then, we compare established optical clearing methods regarding how much signal intensity can be detected in the deep region of tissues, and examine whether the combination of the established clearing methods can improve deep tissue imaging. Finally, automatic whole-tissue quantification for mitotic cells by digital image processing is demonstrated using the embryonic murine epididymal tubule.

Bottom Line: However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed.Through comparison of fixative solutions and of clearing methods, we found optimal conditions to achieve clearer deep-tissue imaging of specific immunolabeled targets and explain what methods result in vivid volume imaging.The procedure for the whole-tissue quantification shown in this article should contribute to systematic measurements of cellular processes in developing organs, accelerating the further understanding of morphogenesis at the single cell level.

View Article: PubMed Central - PubMed

Affiliation: Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

ABSTRACT
Whole-tissue quantification at single-cell resolution has become an inevitable approach for further quantitative understanding of morphogenesis in organ development. The feasibility of the approach has been dramatically increased by recent technological improvements in optical tissue clearing and microscopy. However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed. To provide the appropriate procedure, we here show tips for each critical step of the entire process, including fixation for immunofluorescence, optical clearing, and digital image processing, using developing murine internal organs such as epididymis, kidney, and lung as an example. Through comparison of fixative solutions and of clearing methods, we found optimal conditions to achieve clearer deep-tissue imaging of specific immunolabeled targets and explain what methods result in vivid volume imaging. In addition, we demonstrated that three-dimensional digital image processing after optical clearing produces objective quantitative data for the whole-tissue analysis, focusing on the spatial distribution of mitotic cells in the epididymal tubule. The procedure for the whole-tissue quantification shown in this article should contribute to systematic measurements of cellular processes in developing organs, accelerating the further understanding of morphogenesis at the single cell level.

No MeSH data available.


Related in: MedlinePlus