Fast quantitative time lapse displacement imaging of endothelial cell invasion

Autoři: Christian Steuwe aff001;  Marie-Mo Vaeyens aff002;  Alvaro Jorge-Peñas aff002;  Célie Cokelaere aff001;  Johan Hofkens aff003;  Maarten B. J. Roeffaers aff001;  Hans Van Oosterwyck aff002
Působiště autorů: Centre for Membrane Separations, Adsorption, Catalysis and Spectroscopy for Sustainable Solutions (cMACS), Department of Microbial and Molecular Systems (MS), KU Leuven, Leuven, Belgium aff001;  Biomechanics Section (BMe), Department of Mechanical Engineering, KU Leuven, Leuven, Belgium aff002;  Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Leuven, Belgium aff003;  Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium aff004
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227286


In order to unravel rapid mechano-chemical feedback mechanisms in sprouting angiogenesis, we combine selective plane illumination microscopy (SPIM) and tailored image registration algorithms — further referred to as SPIM-based displacement microscopy — with an in vitro model of angiogenesis. SPIM successfully tackles the problem of imaging large volumes while upholding the spatial resolution required for the analysis of matrix displacements at a subcellular level. Applied to in vitro angiogenic sprouts, this unique methodological combination relates subcellular activity — minute to second time scale growing and retracting of protrusions — of a multicellular systems to the surrounding matrix deformations with an exceptional temporal resolution of 1 minute for a stack with multiple sprouts simultaneously or every 4 seconds for a single sprout, which is 20 times faster than with a conventional confocal setup. Our study reveals collective but non-synchronised, non-continuous activity of adjacent sprouting cells along with correlations between matrix deformations and protrusion dynamics.

Klíčová slova:

Angiogenesis – Collagens – Deformation – Fluorescence imaging – Fluorescence microscopy – Gels – Light – Optical lenses


1. Dembo M, Oliver T. Imaging the Traction Stresses Exerted by Locomoting Cells with the Elastic Substratum Method. 1996;70: 2008–2022.

2. Dembo M, Wang Y. Stresses at the Cell-to-Substrate Interface during Locomotion of Fibroblasts. 1999;76: 2307–2316.

3. Butler J, Fredberg JJ. Traction fields, moments, and strain energy that cells exert on their surroundings. 2002. doi: 10.1152/ajpcell.00270.2001 11832345

4. del Alamo JC, Meili R, Alonso-latorre B, Rodrı J, Juan CA, Aliseda A, et al. Spatio-temporal analysis of eukaryotic cell motility by improved force cytometry. 2007.

5. Legant WR, Miller JS, Blakely BL, Cohen DM, Genin GM, Chen CS. Measurement of mechanical tractions exerted by cells in three-dimensional matrices. Nat Methods. 2010;7: 969–971. doi: 10.1038/nmeth.1531 21076420

6. Steinwachs J, Metzner C, Skodzek K, Lang N, Thievessen I, Mark C, et al. Three-dimensional force microscopy of cells in biopolymer networks. Nat Methods. 2016;13: 171–176. doi: 10.1038/nmeth.3685 26641311

7. Condor M, Steinwachs J, Mark C, Garcia-Aznar JM, Fabry B. Traction Force Microscopy in 3-Dimensional Extracellular Matrix. Curr Protoc Cell Biol. 2017;75: 1–20. doi: 10.1002/cpcb.24 28627753

8. Schwarz US, Soiné JRD. Traction force microscopy on soft elastic substrates: A guide to recent computational advances ☆. BBA—Mol Cell Res. 2015;1853: 3095–3104. doi: 10.1016/j.bbamcr.2015.05.028 26026889

9. Stout DA, Bar-Kochba E, Estrada JB, Toyjanova J, Kesari H, Reichner JS, et al. Mean deformation metrics for quantifying 3D cell–matrix interactions without requiring information about matrix material properties. Proc Natl Acad Sci. 2016;113: 2898–2903. doi: 10.1073/pnas.1510935113 26929377

10. Santi PA. Light sheet fluorescence microscopy: A review. J Histochem Cytochem. 2011;59: 129–138. doi: 10.1369/0022155410394857 21339178

11. Reynaud EG, Peychl J, Huisken J, Tomancak P. Guide to light-sheet microscopy for adventurous biologists. Nat Publ Gr. 2015;12: 30–34. doi: 10.1038/nmeth.3222 25549268

12. Gao L, Shao L, Chen B-C, Betzig E. 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy. Nat Protoc. 2014;9: 1083–1101. doi: 10.1038/nprot.2014.087 24722406

13. Voie A, Burns D, Spelman F. Orthogonal-plane fluorescence optical sectioning: three- dimensional imaging of macroscopic biological specimens. 1993;170: 8371260. doi: 10.1111/j.1365-2818.1993.tb03346.x 8371260

14. Huisken J, Swoger J, Bene F Del, Wittbrodt J, Stelzer EHK. Live Embryos by Selective Plane Illumination Microscopy. 2004;305: 1007–1010.

15. Engelbrecht CJ, Stelzer EHK. Resolution enhancement in a light-sheet-based microscope (SPIM). 2006;31: 1477–1479.

16. Stelzer EHK. Light-sheet fluorescence microscopy for quantitative biology. Nat Methods. 2014;12: 23–26. doi: 10.1038/nmeth.3219 25549266

17. Thorn K, Kellogg D. A quick guide to light microscopy in cell biology. Mol Biol Cell. 2016;27. doi: 10.1091/mbc.E15-02-0088 26768859

18. Mohan K, Purnapatra SB, Mondal PP. Three dimensional fluorescence imaging using multiple light-sheet microscopy. PLoS One. 2014;9: 1–8. doi: 10.1371/journal.pone.0096551 24911061

19. Rasmi CK, Mohan K, Madhangi M, Rajan K, Nongthomba U, Mondal PP. Limited-view light sheet fluorescence microscopy for three dimensional volume imaging. 2017;im. doi: 10.1063/1.4938536

20. Fu Q, Martin BL, Matus DQ, Gao L. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nat Commun. 2016;7. doi: 10.1038/ncomms11088 27004937

21. Verveer PJ, Swoger J, Pampaloni F, Greger K, Marcello M, Stelzer EHK. High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy. Nat Methods. 2007;4: 311–313. doi: 10.1038/nmeth1017 17339847

22. Magidson V, Khodjakov A. Circumventing photodamage in live-cell microscopy. Methods Cell Biol. 2013;114: 1–15. doi: 10.1016/B978-0-12-407761-4.00001-4

23. Udan RS, Piazza VG, Hsu CC -w. C, Hadjantonakis A a.-K, Dickinson ME. Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy. Development. 2014;141: 4406–4414. doi: 10.1242/dev.111021 25344073

24. Patra B, Peng Y-S, Peng C-C, Liao W-H, Chen Y-A, Lin K-H, et al. Migration and vascular lumen formation of endothelial cells in cancer cell spheroids of various sizes. Biomicrofluidics. 2014;8: 052109. doi: 10.1063/1.4895568 25332736

25. Gualda EJ, Simão D, Pinto C, Alves PM, Brito C, Ries J, et al. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy. Front Cell Neurosci. 2014;8: 1–10. doi: 10.3389/fncel.2014.00001

26. Rasmi CK, Madhangi M, Nongthomba U, Pratim Mondal P. Curtailed light sheet microscopy for rapid imaging of macroscopic biological specimens. Microsc Res Tech. 2016;79: 455–458. doi: 10.1002/jemt.22665 27059099

27. Duncan LH, Moyle MW, Shao L, Sengupta T, Ikegami R, Kumar A, et al. Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution. J Vis Exp. 2019; 1–15. doi: 10.3791/59533 31233035

28. Funane T, Hou SS, Zoltowska KM, Van Veluw SJ, Berezovska O, Kumar ATN, et al. Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples. Rev Sci Instrum. 2018;89. doi: 10.1063/1.5018846 29864842

29. Andilla J, Jorand R, Olarte OE, Dufour AC, Cazales M, Montagner YLE, et al. Imaging tissue-mimic with light sheet microscopy: A comparative guideline. Sci Rep. 2017;7: 1–14. doi: 10.1038/s41598-016-0028-x

30. Pampaloni F., Richa R., Ansari N., Stelzer E.H.K. (2015) Live Spheroid Formation Recorded with Light Sheet-Based Fluorescence Microscopy. In: Verveer P. (eds) Advanced Fluorescence Microscopy. Methods in Molecular Biology (Methods and Protocols), vol 1251. Humana Press, New York, NY

31. Schmitz A, Fischer SC, Mattheyer C, Pampaloni F, Stelzer EHK. Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids. Sci Rep. 2017;7: 1–13. doi: 10.1038/s41598-016-0028-x

32. Galland R, Grenci G, Aravind A, Viasnoff V, Studer V, Sibarita J. 3D high- and super- resolution imaging using single-objective SPIM. 2015;12. doi: 10.1038/nmeth.3402 25961414

33. Planchon TA, Liang G, Milkie DE, Davidson MW, Galbraith JA, Galbraith CG, et al. Bessel beam plane illumination. Nat Methods. 2011;8: 417–423. doi: 10.1038/nmeth.1586 21378978

34. Gebhardt JCM. Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells. 2013;10: 421–426. doi: 10.1038/nmeth.2411.Single

35. Kniazeva E, Weidling JW, Singh R, Botvinick EL, Digman MA, Gratton E, et al. Quantification of local matrix deformations and mechanical properties during capillary morphogenesis in 3D. Integr Biol. 2012;4: 431–439. doi: 10.1039/c2ib00120a 22281872

36. Du Y, Herath SCB, Wang Q, Wang D, Asada HH, Chen PCY. Three-Dimensional Characterization of Mechanical Interactions between Endothelial Cells and Extracellular Matrix during Angiogenic Sprouting. Sci Rep. 2016; 21362.

37. Fischer RS, Gardel M, Ma X, Adelstein R, Clare M. Myosin II mediates local cortical tension to guide endothelial cell branching morphogenesis and migration in 3D. Curr Biol. 2010;19: 260–265. doi: 10.1016/j.cub.2008.12.045.Myosin

38. Xue N, Bertulli C, Sadok A, Yan Y, Huang S. Dynamics of filopodium-like protrusion and endothelial cellular motility on one- dimensional extracellular matrix fibrils. 2014.

39. Santos-Oliveira P, Correia A, Rodrigues T, Ribeiro-Rodrigues TM, Matafome P, Rodríguez-Manzaneque JC, et al. The Force at the Tip—Modelling Tension and Proliferation in Sprouting Angiogenesis. PLoS Comput Biol. 2015;11: 1–20. doi: 10.1371/journal.pcbi.1004436 26248210

40. Carmeliet P, De Smet F, Loges S, Mazzone M. Branching morphogenesis and antiangiogenesis candidates: Tip cells lead the way. Nat Rev Clin Oncol. 2009;6: 315–326. doi: 10.1038/nrclinonc.2009.64 19483738

41. Geudens I, Gerhardt O. Coordinating cell behaviour during blood vessel formation. Development. 2011;138: 4569–4583. doi: 10.1242/dev.062323 21965610

42. Bordeleau F, Mason BN, Lollis EM, Mazzola M, Zanotelli MR, Somasegar S, et al. Matrix stiffening promotes a tumor vasculature phenotype. Proc Natl Acad Sci U S A. 2017;114: 492–497. doi: 10.1073/pnas.1613855114 28034921

43. Edgar LT, Underwood CJ, Guilkey JE, Hoying JB, Weiss JA. Extracellular matrix density regulates the rate of neovessel growth and branching in sprouting angiogenesis. PLoS One. 2014;9: 1–10. doi: 10.1371/journal.pone.0085178 24465500

44. Mason BN, Starchenko A, Williams RM, Bonassar LJ, Reinhart-King CA. Tuning three-dimensional collagen matrix stiffness independently of collagen concentration modulates endothelial cell behavior. Acta Biomater. 2013;9: 4635–4644. doi: 10.1016/j.actbio.2012.08.007 22902816

45. Miron-Mendoza M, Seemann J, Grinnell F. The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices. Biomaterials. 2010;31: 6425–6435. doi: 10.1016/j.biomaterials.2010.04.064 20537378

46. Shamloo A, Heilshorn SC. Matrix density mediates polarization and lumen formation of endothelial sprouts in VEGF gradients. Lab Chip. 2010;10: 3061–3068. doi: 10.1039/c005069e 20820484

47. Lee PF, Bai Y, Smith RL, Bayless KJ, Yeh AT. Angiogenic responses are enhanced in mechanically and microscopically characterized, microbial transglutaminase crosslinked collagen matrices with increased stiffness. Acta Biomater. 2013;9: 7178–7190. doi: 10.1016/j.actbio.2013.04.001 23571003

48. Hur SS, Zhao Y, Li Y-S, Botvinick E, Chien S, SUNGSIKH Ur, et al. Live Cells Exert 3-Dimensional Traction Forces on Their Substrata. Cell Mol Bioeng. 2009;2: 425–436. doi: 10.1007/s12195-009-0082-6 19779633

49. Eilken HM, Adams RH. Dynamics of endothelial cell behavior in sprouting angiogenesis. Curr Opin Cell Biol. 2010;22: 617–625. doi: 10.1016/ 20817428

50. Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell. 2011;146: 873–887. doi: 10.1016/j.cell.2011.08.039 21925313

51. Jorge-Peñas A, Bové H, Sanen K, Vaeyens MM, Steuwe C, Roeffaers M, et al. 3D full-field quantification of cell-induced large deformations in fibrillar biomaterials by combining non-rigid image registration with label-free second harmonic generation. Biomaterials. 2017;136: 86–97. doi: 10.1016/j.biomaterials.2017.05.015 28521203

52. Hall MS, Long R, Feng X, Huang YL, Hui CY, Wu M. Toward single cell traction microscopy within 3D collagen matrices. Exp Cell Res. 2013;319: 2396–2408. doi: 10.1016/j.yexcr.2013.06.009 23806281

53. Bayless KJ, Kwak H Il, Su SC. Investigating endothelial invasion and sprouting behavior in three-dimensional collagen matrices. Nat Protoc. 2009;4: 1888–1898. doi: 10.1038/nprot.2009.221 20010936

54. Nguyen D-HTD, Stapleton SC, Yang MT, Cha SS, Choi CK, Galie PA, et al. Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proc Natl Acad Sci U S A. 2013;110: 6712–7. doi: 10.1073/pnas.1221526110 23569284

55. Title B, Title S, Title C, Year C, Holdername C, Author C, et al. Mechanical and Chemical Signaling in Angiogenesis. 2013. doi: 10.1007/s10456-012-9300-2

56. Trappmann B, Baker BM, Polacheck WJ, Choi CK, Burdick JA, Chen CS. Matrix degradability controls multicellularity of 3D cell migration. Nat Commun. 2017;8: 1–8. doi: 10.1038/s41467-016-0009-6

57. Koh W, Stratman AN, Sacharidou A, Davis GE. Chapter 5 In Vitro Three Dimensional Collagen Matrix Models of Endothelial Lumen Formation During Vasculogenesis and Angiogenesis. Methods Enzymol. 2008;443: 83–101. doi: 10.1016/S0076-6879(08)02005-3 18772012

58. Barcellona ML, Cardiel G, Gratton E, Gardiel G, Gratton E. Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides. Biochem Biophys Res Commun. 1990;170: 270–280. doi: 10.1016/0006-291x(90)91270-3 2372293

59. Favilla R, Stecconi G, Cavatorta P, Sartor G, Mazzini A. The interaction of DAPI with phospholipid vesicles and micelles. Biophys Chem. 1993;46: 217–226. doi: 10.1016/0301-4622(93)80015-b 8343569

60. Garcia D. Robust smoothing of gridded data in one and higher dimensions with missing values. Comput Stat Data Anal. 2010;54: 1167–1178. doi: 10.1016/j.csda.2009.09.020 24795488

61. Otsu N. A Threshold Selection Method from Gray-Level Histograms. IEEE Trans Syst Man Cybern. 1979;9: 62–66. doi: 10.1109/TSMC.1979.4310076

62. Klein S, Staring M, Murphy K, Viergever MA, Pluim JPW. Elastix: A toolbox for intensity-based medical image registration. IEEE Trans Med Imaging. 2010;29: 196–205. doi: 10.1109/TMI.2009.2035616 19923044

63. Jorge-Peñas A, Izquierdo-Alvarez A, Aguilar-Cuenca R, Vicente-Manzanares M, Garcia-Aznar JM, Van Oosterwyck H, et al. Free form deformation-based image registration improves accuracy of traction force microscopy. PLoS One. 2015;10: 1–22. doi: 10.1371/journal.pone.0144184 26641883

64. Ubezio B, Blanco RA, Geudens I, Stanchi F, Mathivet T, Jones ML, et al. Synchronization of endothelial Dll4-Notch dynamics switch blood vessels from branching to expansion. Elife. 2016;5: 1–32. doi: 10.7554/eLife.12167 27074663

65. Bentley K, Chakravartula S. The temporal basis of angiogenesis. Philos Trans R Soc B Biol Sci. 2017;372. doi: 10.1098/rstb.2015.0522 28348255

66. Cruys B, Wong BW, Kuchnio A, Verdegem D, Cantelmo AR, Conradi LC, et al. Glycolytic regulation of cell rearrangement in angiogenesis. Nat Commun. 2016;7: 1–15. doi: 10.1038/ncomms12240 27436424

67. Fischer RS, Gardel M, Ma X, Adelstein RS, Waterman CM. Local Cortical Tension by Myosin II Guides 3D Endothelial Cell Branching. Curr Biol. 2009;19: 260–265. doi: 10.1016/j.cub.2008.12.045 19185493

68. Mammoto T, Mammoto A, Ingber DE. Mechanobiology and Developmental Control. Annu Rev Cell Dev Biol. 2013;29: 27–61. doi: 10.1146/annurev-cellbio-101512-122340 24099083

69. Zinn A, Goicoechea SM, Kreider-Letterman G, Maity D, Awadia S, Cedeno-Rosario L, et al. The small GTPase RhoG regulates microtubule-mediated focal adhesion disassembly. Sci Rep. 2019;9: 1–15. doi: 10.1038/s41598-018-37186-2

70. Stehbens S, Wittmann T. Analysis of focal adhesion turnover: A quantitative live cell imaging example. 2015; 335–346. doi: 10.1016/B978-0-12-420138-5.00018-5.Analysis

71. Wolfenson H, Lavelin I, Geiger B. Dynamic Regulation of the Structure and Functions of Integrin Adhesions. 2014;24. doi: 10.1016/j.devcel.2013.02.012.Dynamic

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