OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 3, Iss. 6 — Jun. 1, 2012
  • pp: 1241–1255

Depth-resolved cellular microrheology using HiLo microscopy

Jarett Michaelson, Heejin Choi, Peter So, and Hayden Huang  »View Author Affiliations

Biomedical Optics Express, Vol. 3, Issue 6, pp. 1241-1255 (2012)

View Full Text Article

Enhanced HTML    Acrobat PDF (2033 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



It is increasingly important to measure cell mechanical properties in three-dimensional environments. Particle tracking microrheology (PTM) can measure cellular viscoelastic properties; however, out-of-plane data can introduce artifacts into these measurements. We developed a technique that employs HiLo microscopy to reduce out-of-plane contributions. This method eliminated signals from 90% of probes 0.5 μm or further from the focal plane, while retaining all in-plane probes. We used this technique to characterize live-cell bilayers and found that there were significant, frequency-dependent changes to the extracted cell moduli when compared to conventional analysis. Our results indicate that removal of out-of-plane information is vital for accurate assessments of cell mechanical properties.

© 2012 OSA

OCIS Codes
(170.1530) Medical optics and biotechnology : Cell analysis
(180.0180) Microscopy : Microscopy

ToC Category:
Cell Studies

Original Manuscript: March 21, 2012
Revised Manuscript: April 25, 2012
Manuscript Accepted: April 29, 2012
Published: May 3, 2012

Jarett Michaelson, Heejin Choi, Peter So, and Hayden Huang, "Depth-resolved cellular microrheology using HiLo microscopy," Biomed. Opt. Express 3, 1241-1255 (2012)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. J. Lammerding and R. T. Lee, “The nuclear membrane and mechanotransduction: impaired nuclear mechanics and mechanotransduction in lamin A/C deficient cells,” in Nuclear Organization in Development and Disease, Novartis Foundation Symposium Vol. 264 (Wiley, 2005), pp. 264–273.
  2. T. P. Kole, Y. Tseng, I. Jiang, J. L. Katz, and D. Wirtz, “Intracellular mechanics of migrating fibroblasts,” Mol. Biol. Cell16(1), 328–338 (2005). [CrossRef] [PubMed]
  3. R. G. Wells, “The role of matrix stiffness in regulating cell behavior,” Hepatology47(4), 1394–1400 (2008). [CrossRef] [PubMed]
  4. E. U. Azeloglu, J. Bhattacharya, and K. D. Costa, “Atomic force microscope elastography reveals phenotypic differences in alveolar cell stiffness,” J. Appl. Physiol.105(2), 652–661 (2008). [CrossRef] [PubMed]
  5. D. Fudge, D. Russell, D. Beriault, W. Moore, E. B. Lane, and A. W. Vogl, “The intermediate filament network in cultured human keratinocytes is remarkably extensible and resilient,” PLoS ONE3(6), e2327 (2008). [CrossRef] [PubMed]
  6. H. Huang, A. Asimaki, D. Lo, W. McKenna, and J. Saffitz, “Disparate effects of different mutations in plakoglobin on cell mechanical behavior,” Cell Motil. Cytoskeleton65(12), 964–978 (2008). [CrossRef] [PubMed]
  7. J. Lammerding, P. C. Schulze, T. Takahashi, S. Kozlov, T. Sullivan, R. D. Kamm, C. L. Stewart, and R. T. Lee, “Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction,” J. Clin. Invest.113(3), 370–378 (2004). [PubMed]
  8. D. Wirtz, “Particle-tracking microrheology of living cells: principles and applications,” Annu Rev Biophys38(1), 301–326 (2009). [CrossRef] [PubMed]
  9. J. C. Crocker and B. D. Hoffman, “Multiple-particle tracking and two-point microrheology in cells,” Methods Cell Biol.83, 141–178 (2007). [CrossRef] [PubMed]
  10. M. Jonas, H. Huang, R. D. Kamm, and P. T. So, “Fast fluorescence laser tracking microrheometry, II: quantitative studies of cytoskeletal mechanotransduction,” Biophys. J.95(2), 895–909 (2008). [CrossRef] [PubMed]
  11. A. W. Lau, B. D. Hoffman, A. Davies, J. C. Crocker, and T. C. Lubensky, “Microrheology, stress fluctuations, and active behavior of living cells,” Phys. Rev. Lett.91(19), 198101 (2003). [CrossRef] [PubMed]
  12. C. M. Hale, A. L. Shrestha, S. B. Khatau, P. J. Stewart-Hutchinson, L. Hernandez, C. L. Stewart, D. Hodzic, and D. Wirtz, “Dysfunctional connections between the nucleus and the actin and microtubule networks in laminopathic models,” Biophys. J.95(11), 5462–5475 (2008). [CrossRef] [PubMed]
  13. J. S. Lee, P. Panorchan, C. M. Hale, S. B. Khatau, T. P. Kole, Y. Tseng, and D. Wirtz, “Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiffening response to fluid flow,” J. Cell Sci.119(9), 1760–1768 (2006). [CrossRef] [PubMed]
  14. A. Pai, P. Sundd, and D. F. Tees, “In situ microrheological determination of neutrophil stiffening following adhesion in a model capillary,” Ann. Biomed. Eng.36(4), 596–603 (2008). [CrossRef] [PubMed]
  15. P. Panorchan, J. S. Lee, B. R. Daniels, T. P. Kole, Y. Tseng, and D. Wirtz, “Probing cellular mechanical responses to stimuli using ballistic intracellular nanorheology,” Methods Cell Biol.83, 113–140 (2007). [CrossRef] [PubMed]
  16. P. J. Stewart-Hutchinson, C. M. Hale, D. Wirtz, and D. Hodzic, “Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness,” Exp. Cell Res.314(8), 1892–1905 (2008). [CrossRef] [PubMed]
  17. A. D. van der Meer, Y. Li, M. H. Duits, A. A. Poot, J. Feijen, and I. Vermes, “Shear stress induces a transient and VEGFR-2-dependent decrease in the motion of injected particles in endothelial cells,” Biorheology47(3-4), 179–192 (2010). [PubMed]
  18. Y. Tseng, T. P. Kole, and D. Wirtz, “Micromechanical mapping of live cells by multiple-particle-tracking microrheology,” Biophys. J.83(6), 3162–3176 (2002). [CrossRef] [PubMed]
  19. K. Nishida, M. Yamato, Y. Hayashida, K. Watanabe, K. Yamamoto, E. Adachi, S. Nagai, A. Kikuchi, N. Maeda, H. Watanabe, T. Okano, and Y. Tano, “Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium,” N. Engl. J. Med.351(12), 1187–1196 (2004). [CrossRef] [PubMed]
  20. S. L. Ishaug-Riley, G. M. Crane-Kruger, M. J. Yaszemski, and A. G. Mikos, “Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers,” Biomaterials19(15), 1405–1412 (1998). [CrossRef] [PubMed]
  21. S. L. Ishaug, G. M. Crane, M. J. Miller, A. W. Yasko, M. J. Yaszemski, and A. G. Mikos, “Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds,” J. Biomed. Mater. Res.36(1), 17–28 (1997). [CrossRef] [PubMed]
  22. T. Eschenhagen, C. Fink, U. Remmers, H. Scholz, J. Wattchow, J. Weil, W. Zimmermann, H. H. Dohmen, H. Schäfer, N. Bishopric, T. Wakatsuki, and E. L. Elson, “Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system,” FASEB J.11(8), 683–694 (1997). [PubMed]
  23. M. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett.22(24), 1905–1907 (1997). [CrossRef] [PubMed]
  24. N. Bozinovic, C. Ventalon, T. Ford, and J. Mertz, “Fluorescence endomicroscopy with structured illumination,” Opt. Express16(11), 8016–8025 (2008). [CrossRef] [PubMed]
  25. M. F. Langhorst, J. Schaffer, and B. Goetze, “Structure brings clarity: structured illumination microscopy in cell biology,” Biotechnol. J.4(6), 858–865 (2009). [CrossRef] [PubMed]
  26. S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt.14(3), 030502 (2009). [CrossRef] [PubMed]
  27. D. Lim, K. K. Chu, and J. Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett.33(16), 1819–1821 (2008). [CrossRef] [PubMed]
  28. J. Mertz and J. Kim, “Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection,” J. Biomed. Opt.15(1), 016027 (2010). [CrossRef] [PubMed]
  29. J. C. Crocker and D. G. Grier, “When like charges attract: the effects of geometrical confinement on long-range colloidal interactions,” Phys. Rev. Lett.77(9), 1897–1900 (1996). [CrossRef] [PubMed]
  30. T. G. Mason, “Estimating the viscoelastic moduli of complex fluids using the generalized Stokes-Einstein equation,” Rheologica Acta39(4), 371–378 (2000). [CrossRef]
  31. B. S. Elkin, E. U. Azeloglu, K. D. Costa, and B. Morrison, “Mechanical heterogeneity of the rat hippocampus measured by atomic force microscope indentation,” J. Neurotrauma24(5), 812–822 (2007). [CrossRef] [PubMed]
  32. D. C. Lin, E. K. Dimitriadis, and F. Horkay, “Robust strategies for automated AFM force curve analysis--I. Non-adhesive indentation of soft, inhomogeneous materials,” J. Biomech. Eng.129(3), 430–440 (2007). [CrossRef] [PubMed]
  33. J. Solon, I. Levental, K. Sengupta, P. C. Georges, and P. A. Janmey, “Fibroblast adaptation and stiffness matching to soft elastic substrates,” Biophys. J.93(12), 4453–4461 (2007). [CrossRef] [PubMed]
  34. X. Shi, L. Qin, X. Zhang, K. He, C. Xiong, J. Fang, X. Fang, and Y. Zhang, “Elasticity of cardiac cells on the polymer substrates with different stiffness: an atomic force microscopy study,” Phys. Chem. Chem. Phys.13(16), 7540–7545 (2011). [CrossRef] [PubMed]
  35. L. Cao, A. Wu, and G. A. Truskey, “Biomechanical effects of flow and coculture on human aortic and cord blood-derived endothelial cells,” J. Biomech.44(11), 2150–2157 (2011). [CrossRef] [PubMed]
  36. J. G. McNally, T. Karpova, J. Cooper, and J. A. Conchello, “Three-dimensional imaging by deconvolution microscopy,” Methods19(3), 373–385 (1999). [CrossRef] [PubMed]
  37. A. Ganz, M. Lambert, A. Saez, P. Silberzan, A. Buguin, R. M. Mège, and B. Ladoux, “Traction forces exerted through N-cadherin contacts,” Biol. Cell98(12), 721–730 (2006). [CrossRef] [PubMed]
  38. V. Maruthamuthu, B. Sabass, U. S. Schwarz, and M. L. Gardel, “Cell-ECM traction force modulates endogenous tension at cell-cell contacts,” Proc. Natl. Acad. Sci. U.S.A.108(12), 4708–4713 (2011). [CrossRef] [PubMed]
  39. M. C. DeSantis, S. K. Zareh, X. Li, R. E. Blankenship, and Y. M. Wang, “Single-image axial localization precision analysis for individual fluorophores,” Opt. Express20(3), 3057–3065 (2012). [CrossRef] [PubMed]
  40. P. H. Wu, S. H. Arce, P. R. Burney, and Y. Tseng, “A novel approach to high accuracy of video-based microrheology,” Biophys. J.96(12), 5103–5111 (2009). [CrossRef] [PubMed]
  41. P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience3(4), 237–242 (2004). [CrossRef] [PubMed]
  42. H. P. Kao and A. S. Verkman, “Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position,” Biophys. J.67(3), 1291–1300 (1994). [CrossRef] [PubMed]
  43. V. Levi, Q. Ruan, and E. Gratton, “3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells,” Biophys. J.88(4), 2919–2928 (2005). [CrossRef] [PubMed]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

Supplementary Material

» Media 1: AVI (1962 KB)     
» Media 2: AVI (1962 KB)     

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited