Abstract

Lymph node (LN) is an important immune organ that controls adaptive immune responses against foreign pathogens and abnormal cells. To facilitate efficient immune function, LN has highly organized 3D cellular structures, vascular and lymphatic system. Unfortunately, conventional histological analysis relying on thin-sliced tissue has limitations in 3D cellular analysis due to structural disruption and tissue loss in the processes of fixation and tissue slicing. Optical sectioning confocal microscopy has been utilized to analyze 3D structure of intact LN tissue without physical tissue slicing. However, light scattering within biological tissues limits the imaging depth only to superficial portion of LN cortex. Recently, optical clearing techniques have shown enhancement of imaging depth in various biological tissues, but their efficacy for LN are remained to be investigated. In this work, we established optical clearing procedure for LN and achieved 3D volumetric visualization of the whole cortex of LN. More than 4 times improvement in imaging depth was confirmed by using LN obtained from H2B-GFP/actin-DsRed double reporter transgenic mouse. With adoptive transfer of GFP expressing B cells and DsRed expressing T cells and fluorescent vascular labeling by anti-CD31 and anti-LYVE-1 antibody conjugates, we successfully visualized major cellular-level structures such as T-cell zone, B-cell follicle and germinal center. Further, we visualized the GFP expressing metastatic melanoma cell colony, vasculature and lymphatic vessels in the LN cortex.

© 2015 Optical Society of America

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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  40. Y. Hwang, J. Ahn, J. Mun, S. Bae, Y. U. Jeong, N. A. Vinokurov, and P. Kim, “In vivo analysis of THz wave irradiation induced acute inflammatory response in skin by laser-scanning confocal microscopy,” Opt. Express 22(10), 11465–11475 (2014).
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  41. K. Choe, Y. Hwang, H. Seo, and P. Kim, “In vivo high spatiotemporal resolution visualization of circulating T lymphocytes in high endothelial venules of lymph nodes,” J. Biomed. Opt. 18(3), 036005 (2013).
    [Crossref] [PubMed]

2015 (3)

J. Lian and A. D. Luster, “Chemokine-guided cell positioning in the lymph node orchestrates the generation of adaptive immune responses,” Curr. Opin. Cell Biol. 36, 1–6 (2015).
[Crossref] [PubMed]

D. S. Richardson and J. W. Lichtman, “Clarifying Tissue Clearing,” Cell 162(2), 246–257 (2015).
[Crossref] [PubMed]

H. Seo, Y. Hwang, K. Choe, and P. Kim, “In vivo quantitation of injected circulating tumor cells from great saphenous vein based on video-rate confocal microscopy,” Biomed. Opt. Express 6(6), 2158–2167 (2015).
[Crossref] [PubMed]

2014 (4)

Y. Hwang, J. Ahn, J. Mun, S. Bae, Y. U. Jeong, N. A. Vinokurov, and P. Kim, “In vivo analysis of THz wave irradiation induced acute inflammatory response in skin by laser-scanning confocal microscopy,” Opt. Express 22(10), 11465–11475 (2014).
[Crossref] [PubMed]

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

A. J. Moy, P. C. Lo, and B. Choi, “High-resolution visualization of mouse cardiac microvasculature using optical histology,” Biomed. Opt. Express 5(1), 69–77 (2014).
[Crossref] [PubMed]

M. Nakagawa, R. Schmitz, W. Xiao, C. K. Goldman, W. Xu, Y. Yang, X. Yu, T. A. Waldmann, and L. M. Staudt, “Gain-of-function CCR4 mutations in adult T cell leukemia/lymphoma,” J. Exp. Med. 211(13), 2497–2505 (2014).
[Crossref] [PubMed]

2013 (7)

S. Kwon, G. D. Agollah, G. Wu, W. Chan, and E. M. Sevick-Muraca, “Direct visualization of changes of lymphatic function and drainage pathways in lymph node metastasis of B16F10 melanoma using near-infrared fluorescence imaging,” Biomed. Opt. Express 4(6), 967–977 (2013).
[Crossref] [PubMed]

S. Das, E. Sarrou, S. Podgrabinska, M. Cassella, S. K. Mungamuri, N. Feirt, R. Gordon, C. S. Nagi, Y. Wang, D. Entenberg, J. Condeelis, and M. Skobe, “Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses,” J. Exp. Med. 210(8), 1509–1528 (2013).
[Crossref] [PubMed]

A. J. Moy, M. P. Wiersma, and B. Choi, “Optical histology: a method to visualize microvasculature in thick tissue sections of mouse brain,” PLoS One 8(1), e53753 (2013).
[Crossref] [PubMed]

C. Leahy, H. Radhakrishnan, and V. J. Srinivasan, “Volumetric imaging and quantification of cytoarchitecture and myeloarchitecture with intrinsic scattering contrast,” Biomed. Opt. Express 4(10), 1978–1990 (2013).
[Crossref] [PubMed]

Y. Ding, J. Wang, Z. Fan, D. Wei, R. Shi, Q. Luo, D. Zhu, and X. Wei, “Signal and depth enhancement for in vivo flow cytometer measurement of ear skin by optical clearing agents,” Biomed. Opt. Express 4(11), 2518–2526 (2013).
[Crossref] [PubMed]

C. Yeo, H. Kang, Y. Bae, J. Park, J. S. Nelson, K. J. Lee, and B. Jung, “Development of an Optical Tissue Clearing Laser Probe System,” J. Opt. Soc. Korea 17(4), 289–295 (2013).
[Crossref]

K. Choe, Y. Hwang, H. Seo, and P. Kim, “In vivo high spatiotemporal resolution visualization of circulating T lymphocytes in high endothelial venules of lymph nodes,” J. Biomed. Opt. 18(3), 036005 (2013).
[Crossref] [PubMed]

2012 (9)

H. Peinado, M. Alečković, S. Lavotshkin, I. Matei, B. Costa-Silva, G. Moreno-Bueno, M. Hergueta-Redondo, C. Williams, G. García-Santos, C. Ghajar, A. Nitadori-Hoshino, C. Hoffman, K. Badal, B. A. Garcia, M. K. Callahan, J. Yuan, V. R. Martins, J. Skog, R. N. Kaplan, M. S. Brady, J. D. Wolchok, P. B. Chapman, Y. Kang, J. Bromberg, and D. Lyden, “Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET,” Nat. Med. 18(6), 883–891 (2012).
[Crossref] [PubMed]

K. Becker, N. Jährling, S. Saghafi, R. Weiler, and H.-U. Dodt, “Chemical clearing and dehydration of GFP expressing mouse brains,” PLoS One 7(3), e33916 (2012).
[Crossref] [PubMed]

L. Silvestri, A. Bria, L. Sacconi, G. Iannello, and F. S. Pavone, “Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain,” Opt. Express 20(18), 20582–20598 (2012).
[Crossref] [PubMed]

S. Y. Lee, Q. Chao-Nan, O. A. Seng, C. Peiyi, W. H. Bernice, M. S. Swe, W. J. Chii, H. S. Jacqueline, and S. K. Chee, “Changes in specialized blood vessels in lymph nodes and their role in cancer metastasis,” J. Transl. Med. 10(1), 206 (2012).
[Crossref] [PubMed]

E. Segura, J. Valladeau-Guilemond, M. H. Donnadieu, X. Sastre-Garau, V. Soumelis, and S. Amigorena, “Characterization of resident and migratory dendritic cells in human lymph nodes,” J. Exp. Med. 209(4), 653–660 (2012).
[Crossref] [PubMed]

R. Förster, A. Braun, and T. Worbs, “Lymph node homing of T cells and dendritic cells via afferent lymphatics,” Trends Immunol. 33(6), 271–280 (2012).
[Crossref] [PubMed]

M. Zeng, A. T. Haase, and T. W. Schacker, “Lymphoid tissue structure and HIV-1 infection: life or death for T cells,” Trends Immunol. 33(6), 306–314 (2012).
[Crossref] [PubMed]

K. W. Tan, K. P. Yeo, F. H. Wong, H. Y. Lim, K. L. Khoo, J. P. Abastado, and V. Angeli, “Expansion of cortical and medullary sinuses restrains lymph node hypertrophy during prolonged inflammation,” J. Immunol. 188(8), 4065–4080 (2012).
[Crossref] [PubMed]

H. L. Robinson and R. R. Amara, “Protective immunity from a germinal center sanctuary,” Nat. Med. 18(11), 1614–1616 (2012).
[Crossref] [PubMed]

2011 (5)

K. Kawada and M. M. Taketo, “Significance and mechanism of lymph node metastasis in cancer progression,” Cancer Res. 71(4), 1214–1218 (2011).
[Crossref] [PubMed]

Y. Herishanu, P. Pérez-Galán, D. Liu, A. Biancotto, S. Pittaluga, B. Vire, F. Gibellini, N. Njuguna, E. Lee, L. Stennett, N. Raghavachari, P. Liu, J. P. McCoy, M. Raffeld, M. Stetler-Stevenson, C. Yuan, R. Sherry, D. C. Arthur, I. Maric, T. White, G. E. Marti, P. Munson, W. H. Wilson, and A. Wiestner, “The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia,” Blood 117(2), 563–574 (2011).
[Crossref] [PubMed]

R. P. Kataru, H. Kim, C. Jang, D. K. Choi, B. I. Koh, M. Kim, S. Gollamudi, Y. K. Kim, S. H. Lee, and G. Y. Koh, “T lymphocytes negatively regulate lymph node lymphatic vessel formation,” Immunity 34(1), 96–107 (2011).
[Crossref] [PubMed]

J. L. Hood, R. S. San, and S. A. Wickline, “Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis,” Cancer Res. 71(11), 3792–3801 (2011).
[Crossref] [PubMed]

X. Guo, Z. Guo, H. Wei, H. Yang, Y. He, S. Xie, G. Wu, X. Deng, Q. Zhao, and L. Li, “In vivo comparison of the optical clearing efficacy of optical clearing agents in human skin by quantifying permeability using optical coherence tomography,” Photochem. Photobiol. 87(3), 734–740 (2011).
[Crossref] [PubMed]

2010 (2)

Y. Y. Fu, C. H. Lu, C. W. Lin, J. H. Juang, G. Enikolopov, E. Sibley, A. S. Chiang, and S. C. Tang, “Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution,” J. Biomed. Opt. 15(4), 046018 (2010).
[Crossref] [PubMed]

A. Iwasaki and R. Medzhitov, “Regulation of adaptive immunity by the innate immune system,” Science 327(5963), 291–295 (2010).
[Crossref] [PubMed]

2009 (1)

Y. Y. Fu, C. W. Lin, G. Enikolopov, E. Sibley, A. S. Chiang, and S. C. Tang, “Microtome-free 3-dimensional confocal imaging method for visualization of mouse intestine with subcellular-level resolution,” Gastroenterology 137(2), 453–465 (2009).
[Crossref] [PubMed]

2008 (1)

R. M. Smith, A. Matiukas, C. W. Zemlin, and A. M. Pertsov, “Nondestructive optical determination of fiber organization in intact myocardial wall,” Microsc. Res. Tech. 71(7), 510–516 (2008).
[Crossref] [PubMed]

2007 (3)

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

T. G. Phan, I. Grigorova, T. Okada, and J. G. Cyster, “Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells,” Nat. Immunol. 8(9), 992–1000 (2007).
[Crossref] [PubMed]

M. I. Harrell, B. M. Iritani, and A. Ruddell, “Tumor-induced sentinel lymph node lymphangiogenesis and increased lymph flow precede melanoma metastasis,” Am. J. Pathol. 170(2), 774–786 (2007).
[Crossref] [PubMed]

2006 (3)

S. Hirakawa, L. F. Brown, S. Kodama, K. Paavonen, K. Alitalo, and M. Detmar, “VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites,” Blood 109(3), 1010–1017 (2006).
[Crossref] [PubMed]

H. Qi, J. G. Egen, A. Y. Huang, and R. N. Germain, “Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells,” Science 312(5780), 1672–1676 (2006).
[Crossref] [PubMed]

C. N. Qian, B. Berghuis, G. Tsarfaty, M. Bruch, E. J. Kort, J. Ditlev, I. Tsarfaty, E. Hudson, D. G. Jackson, D. Petillo, J. Chen, J. H. Resau, and B. T. Teh, “Preparing the “soil”: the primary tumor induces vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells,” Cancer Res. 66(21), 10365–10376 (2006).
[Crossref] [PubMed]

2005 (1)

K. A. Soderberg, G. W. Payne, A. Sato, R. Medzhitov, S. S. Segal, and A. Iwasaki, “Innate control of adaptive immunity via remodeling of lymph node feed arteriole,” Proc. Natl. Acad. Sci. U.S.A. 102(45), 16315–16320 (2005).
[Crossref] [PubMed]

2004 (1)

C. A. Murray, W. L. Leong, D. R. McCready, and D. M. Ghazarian, “Histopathological patterns of melanoma metastases in sentinel lymph nodes,” J. Clin. Pathol. 57(1), 64–67 (2004).
[Crossref] [PubMed]

2002 (1)

M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002).
[Crossref] [PubMed]

Abastado, J. P.

K. W. Tan, K. P. Yeo, F. H. Wong, H. Y. Lim, K. L. Khoo, J. P. Abastado, and V. Angeli, “Expansion of cortical and medullary sinuses restrains lymph node hypertrophy during prolonged inflammation,” J. Immunol. 188(8), 4065–4080 (2012).
[Crossref] [PubMed]

Abe, T.

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K. Becker, N. Jährling, S. Saghafi, R. Weiler, and H.-U. Dodt, “Chemical clearing and dehydration of GFP expressing mouse brains,” PLoS One 7(3), e33916 (2012).
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E. Segura, J. Valladeau-Guilemond, M. H. Donnadieu, X. Sastre-Garau, V. Soumelis, and S. Amigorena, “Characterization of resident and migratory dendritic cells in human lymph nodes,” J. Exp. Med. 209(4), 653–660 (2012).
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H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
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H. Qi, J. G. Egen, A. Y. Huang, and R. N. Germain, “Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells,” Science 312(5780), 1672–1676 (2006).
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Y. Y. Fu, C. H. Lu, C. W. Lin, J. H. Juang, G. Enikolopov, E. Sibley, A. S. Chiang, and S. C. Tang, “Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution,” J. Biomed. Opt. 15(4), 046018 (2010).
[Crossref] [PubMed]

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S. Das, E. Sarrou, S. Podgrabinska, M. Cassella, S. K. Mungamuri, N. Feirt, R. Gordon, C. S. Nagi, Y. Wang, D. Entenberg, J. Condeelis, and M. Skobe, “Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses,” J. Exp. Med. 210(8), 1509–1528 (2013).
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S. Das, E. Sarrou, S. Podgrabinska, M. Cassella, S. K. Mungamuri, N. Feirt, R. Gordon, C. S. Nagi, Y. Wang, D. Entenberg, J. Condeelis, and M. Skobe, “Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses,” J. Exp. Med. 210(8), 1509–1528 (2013).
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H. Qi, J. G. Egen, A. Y. Huang, and R. N. Germain, “Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells,” Science 312(5780), 1672–1676 (2006).
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A. Iwasaki and R. Medzhitov, “Regulation of adaptive immunity by the innate immune system,” Science 327(5963), 291–295 (2010).
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Y. Y. Fu, C. W. Lin, G. Enikolopov, E. Sibley, A. S. Chiang, and S. C. Tang, “Microtome-free 3-dimensional confocal imaging method for visualization of mouse intestine with subcellular-level resolution,” Gastroenterology 137(2), 453–465 (2009).
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C. N. Qian, B. Berghuis, G. Tsarfaty, M. Bruch, E. J. Kort, J. Ditlev, I. Tsarfaty, E. Hudson, D. G. Jackson, D. Petillo, J. Chen, J. H. Resau, and B. T. Teh, “Preparing the “soil”: the primary tumor induces vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells,” Cancer Res. 66(21), 10365–10376 (2006).
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R. M. Smith, A. Matiukas, C. W. Zemlin, and A. M. Pertsov, “Nondestructive optical determination of fiber organization in intact myocardial wall,” Microsc. Res. Tech. 71(7), 510–516 (2008).
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M. Zeng, A. T. Haase, and T. W. Schacker, “Lymphoid tissue structure and HIV-1 infection: life or death for T cells,” Trends Immunol. 33(6), 306–314 (2012).
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Biomed. Opt. Express (5)

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S. Hirakawa, L. F. Brown, S. Kodama, K. Paavonen, K. Alitalo, and M. Detmar, “VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites,” Blood 109(3), 1010–1017 (2006).
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J. L. Hood, R. S. San, and S. A. Wickline, “Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis,” Cancer Res. 71(11), 3792–3801 (2011).
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C. N. Qian, B. Berghuis, G. Tsarfaty, M. Bruch, E. J. Kort, J. Ditlev, I. Tsarfaty, E. Hudson, D. G. Jackson, D. Petillo, J. Chen, J. H. Resau, and B. T. Teh, “Preparing the “soil”: the primary tumor induces vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells,” Cancer Res. 66(21), 10365–10376 (2006).
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Cell (2)

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J. Lian and A. D. Luster, “Chemokine-guided cell positioning in the lymph node orchestrates the generation of adaptive immune responses,” Curr. Opin. Cell Biol. 36, 1–6 (2015).
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Y. Y. Fu, C. W. Lin, G. Enikolopov, E. Sibley, A. S. Chiang, and S. C. Tang, “Microtome-free 3-dimensional confocal imaging method for visualization of mouse intestine with subcellular-level resolution,” Gastroenterology 137(2), 453–465 (2009).
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Immunity (1)

R. P. Kataru, H. Kim, C. Jang, D. K. Choi, B. I. Koh, M. Kim, S. Gollamudi, Y. K. Kim, S. H. Lee, and G. Y. Koh, “T lymphocytes negatively regulate lymph node lymphatic vessel formation,” Immunity 34(1), 96–107 (2011).
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K. Choe, Y. Hwang, H. Seo, and P. Kim, “In vivo high spatiotemporal resolution visualization of circulating T lymphocytes in high endothelial venules of lymph nodes,” J. Biomed. Opt. 18(3), 036005 (2013).
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J. Exp. Med. (3)

E. Segura, J. Valladeau-Guilemond, M. H. Donnadieu, X. Sastre-Garau, V. Soumelis, and S. Amigorena, “Characterization of resident and migratory dendritic cells in human lymph nodes,” J. Exp. Med. 209(4), 653–660 (2012).
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S. Das, E. Sarrou, S. Podgrabinska, M. Cassella, S. K. Mungamuri, N. Feirt, R. Gordon, C. S. Nagi, Y. Wang, D. Entenberg, J. Condeelis, and M. Skobe, “Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses,” J. Exp. Med. 210(8), 1509–1528 (2013).
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M. Nakagawa, R. Schmitz, W. Xiao, C. K. Goldman, W. Xu, Y. Yang, X. Yu, T. A. Waldmann, and L. M. Staudt, “Gain-of-function CCR4 mutations in adult T cell leukemia/lymphoma,” J. Exp. Med. 211(13), 2497–2505 (2014).
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J. Immunol. (1)

K. W. Tan, K. P. Yeo, F. H. Wong, H. Y. Lim, K. L. Khoo, J. P. Abastado, and V. Angeli, “Expansion of cortical and medullary sinuses restrains lymph node hypertrophy during prolonged inflammation,” J. Immunol. 188(8), 4065–4080 (2012).
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J. Opt. Soc. Korea (1)

J. Transl. Med. (1)

S. Y. Lee, Q. Chao-Nan, O. A. Seng, C. Peiyi, W. H. Bernice, M. S. Swe, W. J. Chii, H. S. Jacqueline, and S. K. Chee, “Changes in specialized blood vessels in lymph nodes and their role in cancer metastasis,” J. Transl. Med. 10(1), 206 (2012).
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Microsc. Res. Tech. (1)

R. M. Smith, A. Matiukas, C. W. Zemlin, and A. M. Pertsov, “Nondestructive optical determination of fiber organization in intact myocardial wall,” Microsc. Res. Tech. 71(7), 510–516 (2008).
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Nat. Immunol. (1)

T. G. Phan, I. Grigorova, T. Okada, and J. G. Cyster, “Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells,” Nat. Immunol. 8(9), 992–1000 (2007).
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H. L. Robinson and R. R. Amara, “Protective immunity from a germinal center sanctuary,” Nat. Med. 18(11), 1614–1616 (2012).
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H. Peinado, M. Alečković, S. Lavotshkin, I. Matei, B. Costa-Silva, G. Moreno-Bueno, M. Hergueta-Redondo, C. Williams, G. García-Santos, C. Ghajar, A. Nitadori-Hoshino, C. Hoffman, K. Badal, B. A. Garcia, M. K. Callahan, J. Yuan, V. R. Martins, J. Skog, R. N. Kaplan, M. S. Brady, J. D. Wolchok, P. B. Chapman, Y. Kang, J. Bromberg, and D. Lyden, “Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET,” Nat. Med. 18(6), 883–891 (2012).
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Nat. Methods (1)

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
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Opt. Express (2)

Photochem. Photobiol. (1)

X. Guo, Z. Guo, H. Wei, H. Yang, Y. He, S. Xie, G. Wu, X. Deng, Q. Zhao, and L. Li, “In vivo comparison of the optical clearing efficacy of optical clearing agents in human skin by quantifying permeability using optical coherence tomography,” Photochem. Photobiol. 87(3), 734–740 (2011).
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Supplementary Material (6)

NameDescription
» Visualization 1: MOV (7733 KB)      Z-stack movie of non-cleared LN from H2B-GFP/Actin-DsRed mouse
» Visualization 2: MOV (14648 KB)      Z-stack movie of optically cleared LN from H2B-GFP/Actin-DsRed mouse
» Visualization 3: MOV (9390 KB)      Z-stack movie of T/B cell and vascular/lymphatic network in LN
» Visualization 4: MOV (13847 KB)      Z-stack movie of normal LN
» Visualization 5: MOV (12654 KB)      Z-stack movie of metastasized LN at 10 days after the tumor inoculation
» Visualization 6: MOV (12034 KB)      Z-stack movie of metastasized LN at 14 days after the tumor inoculation

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Figures (6)

Fig. 1
Fig. 1

(a) Illustration of optical clearing procedure for LN; Transcardial perfusion with ice-cold PBS and 4% PFA, LN dissection, Fixation and Immersion to optical clearing agents (75% glycerol, Gly; FocusClear, FC). (b) Photograph of lymph node immersed in PBS or FocusClear for 12 hours.

Fig. 2
Fig. 2

Comparison of clearing efficacy of glycerol solution with different (a) concentration and (b) immersion duration. Lymph node dissected from transgenic mouse expressing H2B-GFP (green) in nuclei and actin-DsRed (red) in cytoplasm was imaged. Scale bar, 50 μm.

Fig. 3
Fig. 3

Visualization of intact popliteal lymph node dissected from transgenic mouse expressing H2B-GFP (green) in nuclei and actin-DsRed (red) in cytoplasm. (a) Clearing effect of 75% glycerol and FocusClear. Images were taken at same depth from the surface. Scale bar, 50 μm. (b) XZ projection and (c) 3-Dimensional reconstruction of LN immersed in PBS or FocusClear generated from Z-stack imaging data (Visualization 1 and Visualization 2). Scale bars, 50 μm.

Fig. 4
Fig. 4

Normalized signal intensity of (a) H2B-GFP and (b) actin-DsRed at each depth.

Fig. 5
Fig. 5

Visualization of immune cellular-level structures in inguinal LN showing adoptively transferred lymphocytes; B cells (green), and T cells (red). (a) Representative images of Z-stack imaging data (Visualization 3). Cellular-level structures such as B cell follicle (arrowhead), germinal center (arrow) and T cell zone (asterisk) are distinguishable. (b-c) Mosaic images showing whole inguinal lymph node structure at depth of (b) 65 μm, and (c) 160 μm from the surface. Dashed boxes marked the magnified area. Scale bars, (a) 200 μm, (b-c) 500 μm (mosaic image), 100 μm (magnified image).

Fig. 6
Fig. 6

Visualization of cancer-metastasized LN. (a) Illustration of popliteal LN metastasis model using B16F10-GFP melanoma cells. (b) Photograph of LN with melanoma metastasis immersed in PBS or FocusClear for 12 hours. (c) Serial depth images with 100 μm interval. Distribution of metastasized B16F10-GFP melanoma cells (green), blood vessels (CD31, red) and lymphatic vessels (LYVE-1, blue) are identifiable. (d) Projection and 3D reconstructed images generated by using Z-stack imaging data (Visualization 4, Visualization 5, and Visualization 6) obtained at the area marked as dashed square in Fig. 6(c). (e) Vasculature/lymphatic network around the small-sized colony of GFP expressing cell bodies. Scale bars; (c) 500 μm, (d-e) 100 μm.

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