Abstract
Three-dimensional (3D) cell cultures are important tools in cell biology research and tissue engineering because they more closely resemble the architectural microenvironment of natural tissue, compared to standard two-dimensional cultures. Microscopy techniques that function well for thin, optically transparent cultures, however, are poorly suited for imaging 3D cell cultures. Three-dimensional cultures may be thick and highly scattering, preventing light from penetrating without significant distortion. Techniques that can image thicker biological specimens at high resolution include confocal microscopy, multiphoton microscopy, and optical coherence tomography. In this chapter, these three imaging modalities are described and demonstrated in the assessment of functional and structural features of 3D chitosin scaffolds, 3D micro-topographic substrates from poly-dimethyl siloxane molds, and 3D Matrigel cultures. Using these techniques, dynamic changes to cells in 3D microenvironments can be non-destructively assessed repeatedly over time.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Weaver, V. M., Petersen, O. W., Wang, F., Larabell, C. A., Briand, P., Damsky, C. and Bissell, M. J. (1997) Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J. Cell Biol. 137, 231–245.
Fuchs, E., Tumbar, T. and Guasch, G. (2004) Socializing with the neighbors: stem cells and their niche. Cell 116, 769–778.
Friedrich, M. J. (2003) Studying cancer in 3 dimensions: 3D models foster new insights into tumorigenesis. J. Am. Med. Assoc. 290, 1977–1979.
Lee, J., Cuddihy, M. J. and Kotov, N. A. (2008) Three-dimensional cell culture matrices: state of the art. Tissue Eng. Part B 14, 61–86.
Pawley, J. (ed.) (1995) Handbook of Biological Confocal Microscopy. Springer, New York, NY.
Helmchen, F. and Denk, W. (2005) Deep tissue two-photon microscopy. Nat. Methods 2, 932–940.
Schmitt, J. M. (1999) Optical coherence tomography (OCT): a review. IEEE J. Sel. Top. Quantum Electron. 5, 1205–1215.
Rajadhyaksha, M., Grossman, M., Esterowitz, D., Webb, R. H. and Anderson, R. R., (1995) In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast. J. Invest. Dermatol. 104, 946–952.
Schmitt, J. M., Knüttel, A. and Yadlowsky, M. (1994) Confocal microscopy in turbid media. J. Opt. Soc. Am. A 11, 2226–2235.
Smithpeter, C. L., Dunn, A. K., Welch, A. J. and Richards-Kortum, R. (1998) Penetration depth limits of in vivo confocal reflectance imaging. Appl. Opt. 37, 2749–2754.
Centonze, V. E. and White, J. G. (1998) Multiphoton excitation provides optical sectionsfrom deeper within scattering specimens than confocal imaging. Biophys. J. 75, 2015–2024.
Rubart, M. (2004) Two-photon microscopy of cells and tissue. Circ. Res. 95, 1154–1166.
Huang, D., Swanson, E. A., Lin, C. P., Schuman, J. S., Stinson, W. G., Chang, W., Hee, M. R., Flotte, T., Gregory, K., Puliafito, C. A. and Fujimoto, J. G. (1991) Optical coherence tomography. Science 254, 1178–1181.
Leitgeb, R. Hitzenberger, C. K. and Fercher, A. F. (2003) Performance of Fourier-domain vs. time-domain optical coherence tomography. Opt. Express 11, 889–894.
Nassif, N., Cense, B., Park, B. H., Yun, S. H., Chen, T. C., Bouma, B. E., Tearney, G. J. and de Boer, J. F. (2004) In vivo human retinal imaging by ultrahigh-speed spectral domain optical coherence tomography. Opt. Lett. 29, 480–482.
Huang, S. W., Aguirre, A. D., Huber, R. A., Adler, D. C. and Fujimoto, J. G. (2007) Swept source optical coherence microscopy using a Fourier domain mode-locked laser. Opt. Express 15, 6210–6217.
Welzel, J. (2008) Optical coherence tomography in dermatology: a review. Skin Res. Tech. 7, 1–9.
Aguirre, A. D., Hsiung, P., Ko, T. H., Hartl, I. and Fujimoto, J. G. (2003) High-resolution optical coherence microscopy for high-speed, in vivo cellular imaging. Opt. Lett. 28, 2064–2066.
Vinegoni, C., Ralston, T., Tan, W., Luo, W., Marks, D. L. and Boppart, S. A. (2006) Integrated structural and functional optical imaging combining spectral-domain optical coherence and multiphoton microscopy. Appl. Phys. Lett. 88, 053901.
Tang, S., Sun, C. H., Krasieva, T. B., Chen, Z. and Tromberg, B. J. (2007) Imaging subcellular scattering contrast by using combined optical coherence and multiphoton microscopy. Opt. Lett. 32, 503–505.
Tang, S., Krasieva, T. B., Chen, Z. and Tromberg, B. J. (2006) Combined multiphoton microscopy and optical coherence tomography using a 12-fs broadband source. J. Biomed. Opt. 11, 020502.
Beaurepaire, E., Moreaux, L., Amblard, F. and Mertz, J. (1999) Combined scanning optical coherence and two-photon excited fluorescence microscopy. Opt. Lett. 24, 969–971.
Dunkers, J., Cicerone, M. and Washburn, N. (2003) Collinear optical coherence and confocal fluorescence microscopies for tissue engineering. Opt. Express 11, 3074–3079.
Morgner, U., Drexler, W., Kärtner, F. X., Li, X. D., Pitris, C., Ippen, E. P., and Fujimoto, J. G. (2000) Spectroscopic optical coherence tomography. Opt. Lett. 25, 111–113.
Xu, C., Vinegoni, C., Ralston, T., Luo, W., Tan, W. and Boppart, S. (2006) Spectroscopic spectral-domain optical coherence microscopy. Opt. Lett. 31, 1079–1081.
Schmitt, J. (1998) OCT elastography: imaging microscopic deformation and strain of tissue. Opt. Express 3, 199–211.
Liang, X., Oldenburg, A. L. Crecea, V., Chaney, E. J. and Boppart, S. A. (2008) Optical micro-scale mapping of dynamic biomechanical tissue properties. Opt. Express 16, 11052–11065.
Milner, T. E., Srinivas, S., Wang, X., Malekafzali, A., Van Gemort, M. J., Nelson, J. S., Chen, Z. (1997) Noninvasive imaging of in vivo blood flow velocity using optical Doppler tomography. Opt. Lett. 22, 1119–1121.
Tan, W., Sendemir-Urkmez, A., Fahrner, L. J., Jamison, R., Leckband, D. and Boppart, S. A. (2004) Structural and functional optical imaging of three-dimensional engineered tissue development. Tissue Eng., 10, 1747–1756.
Tan, W., Oldenburg, A. L., Norman, J. J., Desai, T. A. and Boppart, S. A. (2006) Optical coherence tomography of cell dynamics in three-dimensional tissue models. Opt. Express 14, 7159–7171.
Tan, W., Vinegoni, C., Norman, J. J., Desai, T. A. and Boppart, S. A. (2007) Imaging cellular responses to mechanical stimuli within three-dimensional tissue constructs. Microsc. Res. Tech. 70, 361–371
Squirrell, J. M., Wokosin, D. L., White, J. G. and Bavister, B. D., (1999) Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nat. Biotechnol. 17, 763–767.
Tirlapur, U. K., König, K., Peuckert, C., Krieg, R. and Halbhuber, K. J. (2001) Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death. Exp. Cell. Res. 263, 88–97.
Sun, C., Chen, C., Chu, S., Tsai, T., Chen, Y. and Lin, B. (2003) Multiharmonic-generation biopsy of skin. Opt. Lett. 28, 2488–2490.
Norman, J. J. and Desai, T. A. (2005) Control of cellular organization in three dimensions using a microfabricated polydimethylsiloxane-collagen composite tissue scaffold. Tissue Eng. 11, 378–386.
Bestvater, F., Spiess, E., Stobrawa, G., Hacker, M., Feurer, T., Porwol, T., Berchner-Pfannschmidt, U., Wotzlaw, C. and Acker, H. (2002) Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging. J. Microsc. 208, 108–115.
Zipfel, W. R., Williams, R. M., Christie, R., Nikitin, A. Y., Hyman, B. T. and Webb, W. W. (2003) Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. PNAS 100, 7075–7080.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Graf, B.W., Boppart, S.A. (2010). Imaging and Analysis of Three-Dimensional Cell Culture Models. In: Papkovsky, D. (eds) Live Cell Imaging. Methods in Molecular Biology, vol 591. Humana Press. https://doi.org/10.1007/978-1-60761-404-3_13
Download citation
DOI: https://doi.org/10.1007/978-1-60761-404-3_13
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-403-6
Online ISBN: 978-1-60761-404-3
eBook Packages: Springer Protocols