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Two-Photon Intravital Microscopy Animal Preparation Protocol to Study Cellular Dynamics in Pathogenesis

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Light Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1563))

Abstract

Two-photon intravital microscopy (2P-IVM) is an advanced imaging platform that allows the visualization of dynamic processes at subcellular resolution in vivo. Dynamic processes like cell migration, cell proliferation, cell–cell interactions, and cell signaling have an interactive character and occur in complex environments. Hence, it is of pivotal importance to study these processes in living animals, using for example 2P-IVM. 2P-IVM can be performed on a variety of tissues, from the skin of the animal to internal organs, and a variety of methods can be utilized to perform 2P-IVM on these tissues. Here, we discuss the protocols and considerations for four of those 2P-IVM methods, namely tissue explant imaging, skin imaging, surgical exposure imaging, and multi-day window imaging. We carefully compare and explain in depth how to set up each method. Lastly, in the notes section we mention some alternative solutions for the 2P-IVM methods described. In conclusion, this protocol can be used as a guide towards deciding which 2P-IVM method to use and to enable the setup of this method.

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References

  1. Condeelis J, Weissleder R (2010) In vivo imaging in cancer. Cold Spring Harb Perspect Biol 2:a003848. doi:10.1101/cshperspect.a003848

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ritsma L, Ponsioen B, Rheenen J (2012) Intravital imaging of cell signaling in mice. IntraVital 1:2–10. doi:10.4161/intv.20802

    Article  Google Scholar 

  3. Denk W, Strickler J, Webb W (1990) Two-photon laser scanning fluorescence microscopy. Science 248(80):73–76

    Article  CAS  PubMed  Google Scholar 

  4. Campagnola PJ, Millard AC, Terasaki M et al (2002) Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys J 82:493–508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Freund I, Deutsch M, Sprecher A (1986) Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon. Biophys J 50:693–712

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Fein MR, Egeblad M (2013) Caught in the act: revealing the metastatic process by live imaging. Dis Model Mech 6:580–593. doi:10.1242/dmm.009282

    Article  PubMed  PubMed Central  Google Scholar 

  7. Lämmermann T, Germain RN (2014) The multiple faces of leukocyte interstitial migration. Semin Immunopathol 36:227–251. doi:10.1007/s00281-014-0418-8

    Article  PubMed  PubMed Central  Google Scholar 

  8. Kedrin D, Gligorijevic B, Wyckoff J et al (2008) Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods 5:1019–1021

    Article  PubMed  PubMed Central  Google Scholar 

  9. Victora GD, Schwickert TA, Fooksman DR et al (2010) Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143:592–605

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chtanova T, Hampton HR, Waterhouse L A et al (2014) Real-time interactive two-photon photoconversion of recirculating lymphocytes for discontinuous cell tracking in live adult mice. J Biophotonics 7:425–433. doi:10.1002/jbio.201200175

    Article  CAS  PubMed  Google Scholar 

  11. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674

    Article  CAS  PubMed  Google Scholar 

  12. Alexander S, Weigelin B, Winkler F, Friedl P (2013) Preclinical intravital microscopy of the tumour-stroma interface: invasion, metastasis, and therapy response. Curr Opin Cell Biol 25:659–671. doi:10.1016/j.ceb.2013.07.001

    Article  CAS  PubMed  Google Scholar 

  13. Orth JD, Kohler RH, oijer F F et al (2011) Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics. Cancer Res 71:4608–4616. doi:10.1158/0008-5472.CAN-11-0412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rompolas P, Deschene ER, Zito G et al (2012) Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration. Nature 487:496–499. doi:10.1038/nature11218

    Article  PubMed  PubMed Central  Google Scholar 

  15. Chittajallu DR, Florian S, Kohler RH et al (2015) In vivo cell-cycle profiling in xenograft tumors by quantitative intravital microscopy. Nat Methods 12:577–585. doi:10.1038/nmeth.3363

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ritsma L, Ellenbroek SIJ, Zomer A et al (2014) Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging. Nature 507:362–365. doi:10.1038/nature12972

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Coffey SE, Giedt RJ, Weissleder R (2013) Automated analysis of clonal cancer cells by intravital imaging. IntraVital. doi:10.4161/intv.26138

    PubMed  PubMed Central  Google Scholar 

  18. Zomer A, Inge Johanna Ellenbroek S, Ritsma L et al (2013) Brief report: intravital imaging of cancer stem cell plasticity in mammary tumors. Stem Cells 31:602–606. doi:10.1002/stem.1296

    Article  CAS  PubMed  Google Scholar 

  19. Mempel TR, Henrickson SE, Andrian UH (2004) T-cell priming by dendriticcells in lymph nodes occurs in three distinct phases. Nature 427:154–159

    Article  CAS  PubMed  Google Scholar 

  20. Bousso P (2008) T-cell activation by dendritic cells in the lymph node: lessons from the movies. Nat Rev Immunol 8:675–684. doi:10.1038/nri2379

    Article  CAS  PubMed  Google Scholar 

  21. Shakhar G, Lindquist RL, Skokos D et al (2005) Stable T cell-dendritic cell interactions precede the development of both tolerance and immunity in vivo. Nat Immunol 6:707–714. doi:10.1038/ni1210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Germain RN, Robey EA, Cahalan MD (2012) A decade of imaging cellular motility and interaction dynamics in the immune system. Science 336:1676–1681. doi:10.1126/science.1221063

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lee W-Y, Sanz M-J, Wong CHY et al (2014) Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia. Proc Natl Acad Sci U S A 111:13936–13941

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Tanaka K, Toiyama Y, Okugawa Y et al (2014) In vivo optical imaging of cancer metastasis using multiphoton microscopy: a short review. Am J Transl Res 6:179–187

    PubMed  PubMed Central  Google Scholar 

  25. Zal T, Chodaczek G (2010) Intravital imaging of anti-tumor immune response and the tumor microenvironment. Semin Immunopathol 32:305–317. doi:10.1007/s00281-010-0217-9

    Article  PubMed  PubMed Central  Google Scholar 

  26. Phan TG, Bullen A (2010) Practical intravital two-photon microscopy for immunological research: faster, brighter, deeper. Immunol Cell Biol 88:438–444. doi:10.1038/icb.2009.116

    Article  PubMed  Google Scholar 

  27. ank M M, Santos AF, Direnberger S et al (2008) A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat Methods 5:805–811

    Article  Google Scholar 

  28. Bogdanov AA, Lin CP, Simonova M et al Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment. Neoplasia 4:228–236

    Google Scholar 

  29. Timpson P, McGhee EJ, Morton JP et al (2011) Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53. Cancer Res 71:747–757

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Janssen A, Beerling E, Medema R, Rheenen J (2013) Intravital FRET imaging of tumor cell viability and mitosis during chemotherapy. PLoS One 8:e64029. doi:10.1371/journal.pone.0064029

    Article  PubMed  PubMed Central  Google Scholar 

  31. Nobis M, McGhee EJ, Morton JP et al (2013) Intravital FLIM-FRET imaging reveals dasatinib-induced spatial control of src in pancreatic cancer. Cancer Res 73:4674–4686. doi:10.1158/0008-5472.CAN-12-4545

    Article  CAS  PubMed  Google Scholar 

  32. Förster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Phys 437:55–75

    Article  Google Scholar 

  33. Hochreiter B, Garcia AP, Schmid JA (2015) Fluorescent proteins as genetically encoded FRET biosensors in life sciences. Sensors 15:26281–26314. doi:10.3390/s151026281

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lakowicz JR (2006) Principles of Fluorescence Spectroscopy, 3rd edition, third edit. Springer

    Google Scholar 

  35. Burford JL, Villanueva K, Lam L et al (2014) Intravital imaging of podocyte calcium in glomerular injury and disease. J Clin Invest 124:2050–2058. doi:10.1172/JCI71702

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Qi H, Egen JG, Huang AYC, Germain RN (2006) Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312(80):1672–1676. doi:10.1126/science.1125703

    Article  CAS  PubMed  Google Scholar 

  37. Conway JRW, Carragher NO, Timpson P (2014) Developments in preclinical cancer imaging: innovating the discovery of therapeutics. Nat Rev Cancer 14:314–328. doi:10.1038/nrc3724

    Article  CAS  PubMed  Google Scholar 

  38. Prunier C, Josserand V, Vollaire J, et al. (2016) LIM kinase inhibitor pyr1 reduces the growth and metastatic load of breast cancers. Cancer Res 0008–5472.CAN–15–1864–. doi: 10.1158/0008-5472.CAN-15-1864

    Google Scholar 

  39. Budin G, Yang KS, Reiner T, Weissleder R (2011) Bioorthogonal probes for polo-like kinase 1 imaging and quantification. Angew Chem Int Ed Engl 50:9378–9381. doi:10.1002/anie.201103273

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Reiner T, Earley S, Turetsky A, Weissleder R (2010) Bioorthogonal small-molecule ligands for PARP1 imaging in living cells. Chembiochem 11:2374–2377. doi:10.1002/cbic.201000477

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Laughney AM, Kim E, Sprachman MM et al (2014) Single-cell pharmacokinetic imaging reveals a therapeutic strategy to overcome drug resistance to the microtubule inhibitor eribulin. Sci Transl Med 6:261ra152. doi:10.1126/scitranslmed.3009318

    Article  PubMed  PubMed Central  Google Scholar 

  42. Hirata E, Girotti MR, Viros A et al (2015) Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK signaling. Cancer Cell 27:574–588. doi:10.1016/j.ccell.2015.03.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Dzhagalov IL, Melichar HJ, Ross JO, et al. (2012) Two-photon imaging of the immune system. Curr Protoc Cytom Chapter 12:Unit12.26. doi: 10.1002/0471142956.cy1226s60

    Google Scholar 

  44. Kerschensteiner M, Reuter MS, Lichtman JW, Misgeld T (2008) Ex vivo imaging of motor axon dynamics in murine triangularis sterni explants. Nat Protoc 3:1645–1653

    Article  PubMed  PubMed Central  Google Scholar 

  45. Li JL, Goh CC, Keeble JL et al (2012) Intravital multiphoton imaging of immune responses in the mouse ear skin. Nat Protoc 7:221–234. doi:10.1038/nprot.2011.438

    Article  CAS  PubMed  Google Scholar 

  46. Donndorf P, Ludwig M, Wildschütz F et al. (2013) Intravital microscopy of the microcirculation in the mouse cremaster muscle for the analysis of peripheral stem cell migration. J Vis Exp e50485

    Google Scholar 

  47. Masedunskas A, Porat-Shliom N, Tora M et al. (2013) Intravital microscopy for imaging subcellular structures in live mice expressing fluorescent proteins. J Vis Exp e50558

    Google Scholar 

  48. Ewald AJ, Werb Z, Egeblad M (2011) Preparation of mice for long-term intravital imaging of the mammary gland. Cold Spring Harb Protoc 2011:pdb.prot5562. doi: 10.1101/pdb.prot5562

    Google Scholar 

  49. Liou HLR, Myers JT, Barkauskas DS, Huang AY (2012) Intravital imaging of the mouse popliteal lymph node. J Vis Exp e3720

    Google Scholar 

  50. Sellers SL, Payne GW (2011) Intravital microscopy of the inguinal lymph node. J Vis Exp e2551

    Google Scholar 

  51. Ritsma L, Steller EJ a, Ellenbroek SIJ, et al. (2013) Surgical implantation of an abdominal imaging window for intravital microscopy. Nat Protoc 8:583–594. doi: 10.1038/nprot.2013.026

  52. Palmer GM, Fontanella AN, Shan S et al (2011) In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters. Nat Protoc 6:1355–1366

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Yang G, Pan F, Parkhurst CN et al (2010) Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat Protoc 5:201–208

    Article  PubMed  PubMed Central  Google Scholar 

  54. Holtmaat A, Bonhoeffer T, Chow DK et al (2009) Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc 4:1128–1144. doi:10.1038/nprot.2009.89

    Article  PubMed  PubMed Central  Google Scholar 

  55. Alieva M, Ritsma L, Giedt RJ et al (2014) Imaging windows for long-term intravital imaging: General overview and technical insights. IntraVital 3:e29917. doi:10.4161/intv.29917

    Article  PubMed  PubMed Central  Google Scholar 

  56. Farrar MJ, Schaffer CB (2014) A procedure for implanting a spinal chamber for longitudinal in vivo imaging of the mouse spinal cord. J Vis Exp e52196. doi:10.3791/52196

  57. Torabi-Parizi P, Vrisekoop N, Kastenmuller W et al (2014) Pathogen-related differences in the abundance of presented antigen are reflected in CD4+ T cell dynamic behavior and effector function in the lung. J Immunol 192:1651–1660

    Article  PubMed  PubMed Central  Google Scholar 

  58. Chan KT, Jones SW, Brighton HE et al (2014) Intravital imaging of a spheroid-based orthotopic model of melanoma in the mouse ear skin. IntraVital 2:e25805. doi:10.4161/intv.25805

    Article  Google Scholar 

  59. Pineda CM, Park S, Mesa KR et al (2015) Intravital imaging of hair follicle regeneration in the mouse. Nat Protoc 10:1116–1130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Peters NC, Egen JG, Secundino N et al (2008) In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 321:970–974. doi:10.1126/science.1159194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Ariotti S, Beltman JB, Chodaczek G et al (2012) Tissue-resident memory CD8+ T cells continuously patrol skin epithelia to quickly recognize local antigen. Proc Natl Acad Sci U S A 109:19739–19744

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Débarre D, Olivier N, Supatto W, Beaurepaire E (2014) Mitigating phototoxicity during multiphoton microscopy of live Drosophila embryos in the 1.0-1.2 μm wavelength range. PLoS One 9:e104250. doi:10.1371/journal.pone.0104250

    Article  PubMed  PubMed Central  Google Scholar 

  63. Ritsma L, Vrisekoop N, Rheenen J (2013) In vivo imaging and histochemistry are combined in the cryosection labelling and intravital microscopy technique. Nat Commun 4:2366. doi:10.1038/ncomms3366

    Article  PubMed  Google Scholar 

  64. Verhoeven D, Teijaro JR, Farber DL (2009) Pulse-oximetry accurately predicts lung pathology and the immune response during influenza infection. Virology 390:151–156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Yuryev M, Molotkov D, Khiroug L (2014) In vivo two-photon microscopy of single nerve endings in skin. J Vis Exp e51045

    Google Scholar 

  66. Bochner F, Fellus-Alyagor L, Kalchenko V et al (2015) A novel intravital imaging window for longitudinal microscopy of the mouse ovary. Sci Rep 5:12446

    Article  PubMed  PubMed Central  Google Scholar 

  67. Stoll S, Delon J, Brotz TM, Germain RN (2002) Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science 296:1873–1876

    Article  PubMed  Google Scholar 

  68. Miller MJ, Wei SH, Parker I, Cahalan MD (2002) Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296:1869–1873

    Article  CAS  PubMed  Google Scholar 

  69. Bousso P, Robey E (2003) Dynamics of CD8+ T cell priming by dendritic cells in intact lymph nodes. Nat Immunol 4:579–585

    Article  CAS  PubMed  Google Scholar 

  70. Lämmermann T, Afonso PV, Angermann BR et al (2013) Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo. Nature 498:371–375. doi:10.1038/nature12175

    Article  PubMed  Google Scholar 

  71. Patsialou A, Bravo-Cordero JJ, Wang Y et al. (2013) Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors IntraVital 2:e25294

    Google Scholar 

  72. Zomer A, Maynard C, Verweij FJ et al (2015) In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior. Cell 161:1046–1057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Nakasone ES, Askautrud HA, Kees T et al (2012) Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance. Cancer Cell 21:488–503. doi:10.1016/j.ccr.2012.02.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Engelhardt JJ, oldajipour B B, Beemiller P et al (2012) Marginating dendritic cells of the tumor microenvironment cross-present tumor antigens and stably engage tumor-specific T cells. Cancer Cell 21:402–417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Arnon TI, Horton RM, Grigorova IL, Cyster JG (2013) Visualization of splenic marginal zone B-cell shuttling and follicular B-cell egress. Nature 493:684–688

    Article  CAS  PubMed  Google Scholar 

  76. Ritsma L, Steller EJA, Beerling E, et al. (2012) Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis. Sci Transl Med 4: 158ra145–158ra145

    Google Scholar 

  77. Beerling E, Seinstra D, de Wit E, et al. Plasticity between epithelial and mesenchymal states unlinks emt from metastasis-enhancing stem cell capacity. Cell Rep. doi: 10.1016/j.celrep.2016.02.034

    Google Scholar 

  78. Manning CS, Jenkins R, Hooper S, et al. (2013) Intravital imaging reveals conversion between distinct tumor vascular morphologies and localized vascular response to Sunitinib. pp 1–12.

    Google Scholar 

  79. Lai CP, Kim EY, Badr CE et al (2015) Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters. Nat Commun 6:7029. doi:10.1038/ncomms8029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Snuderl M, Batista A, Kirkpatrick ND et al (2013) Targeting placental growth factor/neuropilin 1 pathway inhibits growth and spread of medulloblastoma. Cell 152:1065–1076. doi:10.1016/j.cell.2013.01.036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We apologize in advance to those authors whose contributions are omitted due to space restrictions. L.R. was supported by a Rubicon grant from the Netherlands Organization for Scientific Research (NWO: 825.13.016), a postdoctoral fellowship from the Susan G. Komen foundation (PDF15329694), and a Gisela Thier Fellowship from the Leiden University Medical Center (LUMC).

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    Authors and Affiliations

    1. Department of Respiratory Medicine, Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands

      Erinke van Grinsven & Nienke Vrisekoop

    2. Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands

      Chloé Prunier & Laila Ritsma

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    1. Erinke van Grinsven
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    2. Chloé Prunier
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    3. Nienke Vrisekoop
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    4. Laila Ritsma
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    Correspondence to Laila Ritsma .

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    Editors and Affiliations

    1. Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California, USA

      Yolanda Markaki

    2. Biozentrum der LMU Munchen, Planegg-Martinsried, Germany

      Hartmann Harz

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    van Grinsven, E., Prunier, C., Vrisekoop, N., Ritsma, L. (2017). Two-Photon Intravital Microscopy Animal Preparation Protocol to Study Cellular Dynamics in Pathogenesis. In: Markaki, Y., Harz, H. (eds) Light Microscopy. Methods in Molecular Biology, vol 1563. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6810-7_4

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    • DOI: https://doi.org/10.1007/978-1-4939-6810-7_4

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    • Print ISBN: 978-1-4939-6808-4

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