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

  • Erinke van Grinsven
  • Chloé Prunier
  • Nienke Vrisekoop
  • Laila RitsmaEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Two-photon intravital microscopy Explant Skin Surgical exposure Imaging window 

Notes

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).

References

  1. 1.
    Condeelis J, Weissleder R (2010) In vivo imaging in cancer. Cold Spring Harb Perspect Biol 2:a003848. doi: 10.1101/cshperspect.a003848 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ritsma L, Ponsioen B, Rheenen J (2012) Intravital imaging of cell signaling in mice. IntraVital 1:2–10. doi: 10.4161/intv.20802 CrossRefGoogle Scholar
  3. 3.
    Denk W, Strickler J, Webb W (1990) Two-photon laser scanning fluorescence microscopy. Science 248(80):73–76CrossRefPubMedGoogle Scholar
  4. 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 82:493–508CrossRefPubMedPubMedCentralGoogle Scholar
  5. 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 50:693–712CrossRefPubMedPubMedCentralGoogle Scholar
  6. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kedrin D, Gligorijevic B, Wyckoff J et al (2008) Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods 5:1019–1021CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Victora GD, Schwickert TA, Fooksman DR et al (2010) Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143:592–605CrossRefPubMedPubMedCentralGoogle Scholar
  10. 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 CrossRefPubMedGoogle Scholar
  11. 11.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674CrossRefPubMedGoogle Scholar
  12. 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 CrossRefPubMedGoogle Scholar
  13. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Coffey SE, Giedt RJ, Weissleder R (2013) Automated analysis of clonal cancer cells by intravital imaging. IntraVital. doi: 10.4161/intv.26138 PubMedPubMedCentralGoogle Scholar
  18. 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 CrossRefPubMedGoogle Scholar
  19. 19.
    Mempel TR, Henrickson SE, Andrian UH (2004) T-cell priming by dendriticcells in lymph nodes occurs in three distinct phases. Nature 427:154–159CrossRefPubMedGoogle Scholar
  20. 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 CrossRefPubMedGoogle Scholar
  21. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 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 111:13936–13941CrossRefPubMedPubMedCentralGoogle Scholar
  24. 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 6:179–187PubMedPubMedCentralGoogle Scholar
  25. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 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 CrossRefPubMedGoogle Scholar
  27. 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 5:805–811CrossRefGoogle Scholar
  28. 28.
    Bogdanov AA, Lin CP, Simonova M et al Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment. Neoplasia 4:228–236Google Scholar
  29. 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 71:747–757CrossRefPubMedPubMedCentralGoogle Scholar
  30. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 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 CrossRefPubMedGoogle Scholar
  32. 32.
    Förster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Phys 437:55–75CrossRefGoogle Scholar
  33. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Lakowicz JR (2006) Principles of Fluorescence Spectroscopy, 3rd edition, third edit. SpringerGoogle Scholar
  35. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 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 CrossRefPubMedGoogle Scholar
  37. 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 CrossRefPubMedGoogle Scholar
  38. 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-15-1864Google Scholar
  39. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 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.cy1226s60Google Scholar
  44. 44.
    Kerschensteiner M, Reuter MS, Lichtman JW, Misgeld T (2008) Ex vivo imaging of motor axon dynamics in murine triangularis sterni explants. Nat Protoc 3:1645–1653CrossRefPubMedPubMedCentralGoogle Scholar
  45. 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 CrossRefPubMedGoogle Scholar
  46. 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 e50485Google Scholar
  47. 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 e50558Google Scholar
  48. 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.prot5562Google Scholar
  49. 49.
    Liou HLR, Myers JT, Barkauskas DS, Huang AY (2012) Intravital imaging of the mouse popliteal lymph node. J Vis Exp e3720Google Scholar
  50. 50.
    Sellers SL, Payne GW (2011) Intravital microscopy of the inguinal lymph node. J Vis Exp e2551Google Scholar
  51. 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. 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 6:1355–1366CrossRefPubMedPubMedCentralGoogle Scholar
  53. 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 5:201–208CrossRefPubMedPubMedCentralGoogle Scholar
  54. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  55. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  56. 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. 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 192:1651–1660CrossRefPubMedPubMedCentralGoogle Scholar
  58. 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 CrossRefGoogle Scholar
  59. 59.
    Pineda CM, Park S, Mesa KR et al (2015) Intravital imaging of hair follicle regeneration in the mouse. Nat Protoc 10:1116–1130CrossRefPubMedPubMedCentralGoogle Scholar
  60. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  61. 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 109:19739–19744CrossRefPubMedPubMedCentralGoogle Scholar
  62. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  63. 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 CrossRefPubMedGoogle Scholar
  64. 64.
    Verhoeven D, Teijaro JR, Farber DL (2009) Pulse-oximetry accurately predicts lung pathology and the immune response during influenza infection. Virology 390:151–156CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Yuryev M, Molotkov D, Khiroug L (2014) In vivo two-photon microscopy of single nerve endings in skin. J Vis Exp e51045Google Scholar
  66. 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 5:12446CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Stoll S, Delon J, Brotz TM, Germain RN (2002) Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science 296:1873–1876CrossRefPubMedGoogle Scholar
  68. 68.
    Miller MJ, Wei SH, Parker I, Cahalan MD (2002) Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296:1869–1873CrossRefPubMedGoogle Scholar
  69. 69.
    Bousso P, Robey E (2003) Dynamics of CD8+ T cell priming by dendritic cells in intact lymph nodes. Nat Immunol 4:579–585CrossRefPubMedGoogle Scholar
  70. 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 CrossRefPubMedGoogle Scholar
  71. 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:e25294Google Scholar
  72. 72.
    Zomer A, Maynard C, Verweij FJ et al (2015) In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior. Cell 161:1046–1057CrossRefPubMedPubMedCentralGoogle Scholar
  73. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  74. 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 21:402–417CrossRefPubMedPubMedCentralGoogle Scholar
  75. 75.
    Arnon TI, Horton RM, Grigorova IL, Cyster JG (2013) Visualization of splenic marginal zone B-cell shuttling and follicular B-cell egress. Nature 493:684–688CrossRefPubMedGoogle Scholar
  76. 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: 158ra145–158ra145Google Scholar
  77. 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.2016.02.034Google Scholar
  78. 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. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  80. 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 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Erinke van Grinsven
    • 1
  • Chloé Prunier
    • 2
  • Nienke Vrisekoop
    • 1
  • Laila Ritsma
    • 2
    Email author
  1. 1.Department of Respiratory Medicine, Laboratory of Translational ImmunologyUniversity Medical Center UtrechtUtrechtThe Netherlands
  2. 2.Department of Molecular Cell BiologyLeiden University Medical CenterLeidenThe Netherlands

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