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Intravital Confocal and Two-Photon Imaging of Dual-Color Cells and Extracellular Matrix Mimics

Published online by Cambridge University Press:  04 February 2013

Ufuk Bal ,
Volker Andresen ,
Brenda Baggett  and
Urs Utzinger
Ufuk Bal
Affiliation:
Ege University, Electrical and Electronics Engineering Department, Izmir 35100, Turkey
Volker Andresen
Affiliation:
LaVision BioTec GmbH, Astastrasse 14, Bielefeld, D-33617, Germany
Brenda Baggett
Affiliation:
Biomedical Engineering, University of Arizona, 1127 E James E. Rogers Way, Tucson, AZ 85721, USA
Urs Utzinger*
Affiliation:
Biomedical Engineering, University of Arizona, 1127 E James E. Rogers Way, Tucson, AZ 85721, USA
*
*Corresponding author. E-mail: utzinger@u.arizona.edu
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Abstract

We report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage.

Keywords

extracellular matrix mimicsconfocal microscopytwo-photon microscopyphotobleachingDsRed2eGFP
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 1 , February 2013 , pp. 201 - 212
Copyright
Copyright © Microscopy Society of America 2013

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References

Andresen, V., Alexander, S., Heupel, W.M., Hirschberg, M., Hoffman, R.M. & Friedl, P. (2009). Infrared multiphoton microscopy: Subcellular-resolved deep tissue imaging. Curr Opin Biotechnol 20(1), 54–62.CrossRefGoogle ScholarPubMed
Atala, A. & Lanza, R.P. (2001). Methods of Tissue Engineering. San Diego, CA: Academic Press.Google ScholarPubMed
Baird, I.S., Yau, A.Y. & Mann, B.K. (2008). Mammalian cell-seeded hydrogel microarrays printed via dip-pin technology. Biotechniques 44(2), 249–256.CrossRefGoogle ScholarPubMed
Bjorkoy, G., Lamark, T., Brech, A., Outzen, H., Perander, M., Overvatn, A., Stenmark, H. & Johansen, T. (2005). p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 171(4), 603–614.CrossRefGoogle Scholar
Blab, G.A., Lommerse, P.H.M., Cognet, L., Harms, G.S. & Schmidt, T. (2001). Two-photon excitation action cross-sections of the autofluorescent proteins. Chem Phys Lett 350(1-2), 71–77.CrossRefGoogle Scholar
Black, L.D. 3rd, Meyers, J.D., Weinbaum, J.S., Shvelidze, Y.A. & Tranquillo, R.T. (2009). Cell-induced alignment augments twitch force in fibrin gel-based engineered myocardium via gap junction modification. Tissue Eng Part A 15(10), 3099–3108.CrossRefGoogle ScholarPubMed
Cao, J., Chiarelli, C., Kozarekar, P. & Adler, H.L. (2005). Membrane type 1-matrix metalloproteinase promotes human prostate cancer invasion and metastasis. Thromb Haemost 93(4), 770–778.Google ScholarPubMed
Cao, Z., Gilbert, R.J. & He, W. (2009). Simple agarose-chitosan gel composite system for enhanced neuronal growth in three dimensions. Biomacromolecules 10(10), 2954–2959.CrossRefGoogle ScholarPubMed
Condeelis, J. & Segall, J.E. (2003). Intravital imaging of cell movement in tumours. Nat Rev Cancer 3(12), 921–930.CrossRefGoogle ScholarPubMed
Denk, W., Strickler, J.H. & Webb, W.W. (1990). Two-photon laser scanning fluorescence microscopy. Science 248(4951), 73–76.CrossRefGoogle ScholarPubMed
Drobizhev, M., Tillo, S., Makarov, N.S., Hughes, T.E. & Rebane, A. (2009). Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins. J Phys Chem B 113(4), 855–859.CrossRefGoogle ScholarPubMed
Durr, N.J., Weisspfennig, C.T., Holfeld, B.A. & Ben-Yakar, A. (2011). Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues. J Biomed Opt 16(2), 026008. CrossRefGoogle ScholarPubMed
Friedl, P. (2004). Dynamic imaging of cellular interactions with extracellular matrix. Histochem Cell Biol 122(3), 183–190.CrossRefGoogle ScholarPubMed
Gelman, R.A., Poppke, D.C. & Piez, K.A. (1979). Collagen fibril formation in vitro. The role of the nonhelical terminal regions. J Biol Chem 254(22), 11741–11745.CrossRefGoogle ScholarPubMed
Goldman, R.D., Swedlow, J. & Spector, D.L. (2009). Live Cell Imaging: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
Griess, G.A., Moreno, E.T., Easom, R.A. & Serwer, P. (1989). The sieving of spheres during agarose gel electrophoresis: Quantitation and modeling. Biopolymers 28(8), 1475–1484.CrossRefGoogle ScholarPubMed
Hadjantonakis, A.K. & Papaioannou, V.E. (2004). Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice. BMC Biotechnol 4, 33.CrossRefGoogle ScholarPubMed
Hartmann, A., Boukamp, P. & Friedl, P. (2006). Confocal reflection imaging of 3D fibrin polymers. Blood Cells Mol Dis 36(2), 191–193.CrossRefGoogle ScholarPubMed
Heikal, A.A., Hess, S.T., Baird, G.S., Tsien, R.Y. & Webb, W.W. (2000). Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: Coral red (dsRed) and yellow (Citrine). Proc Natl Acad Sci USA 97(22), 11996–12001.CrossRefGoogle ScholarPubMed
Hoying, J.B., Boswell, C.A. & Williams, S.K. (1996). Angiogenic potential of microvessel fragments established in three-dimensional collagen gels. In Vitro Cell Dev Biol Anim 32(7), 409–419.CrossRefGoogle ScholarPubMed
Huang, Y., Onyeri, S., Siewe, M., Moshfeghian, A. & Madihally, S.V. (2005). In vitro characterization of chitosan- gelatin scaffolds for tissue engineering. Biomaterials 26(36), 7616–7627.CrossRefGoogle ScholarPubMed
International Commission on Non-ionizing Radiation Protection. (2010). ICNIRP statement—Protection of workers against ultraviolet radiation. Health Phys 99(1), 66–87.CrossRefGoogle Scholar
Jacques, S.L. & Gareau, D.S. (2006). Confocal microscopy to measure tissue optical properties. In Saratov Fall Meeting 2005: Optical Technologies in Biophysics and Medicine VII, pp. 61630X–61635. Cardiff, UK: SPIE.Google Scholar
Jockenhoevel, S., Zund, G., Hoerstrup, S.P., Chalabi, K., Sachweh, J.S., Demircan, L., Messmer, B.J. & Turina, M. (2001). Fibrin gel—Advantages of a new scaffold in cardiovascular tissue engineering. Eur J Cardiothorac Surg 19(4), 424–430.CrossRefGoogle ScholarPubMed
Kim, Y., Comte, I., Szabo, G., Hockberger, P. & Szele, F.G. (2009). Adult mouse subventricular zone stem and progenitor cells are sessile and epidermal growth factor receptor negatively regulates neuroblast migration. PLoS One 4(12), e8122. CrossRefGoogle ScholarPubMed
Kobat, D., Durst, M.E., Nishimura, N., Wong, A.W., Schaffer, C.B. & Xu, C. (2009). Deep tissue multiphoton microscopy using longer wavelength excitation. Opt Express 17(16), 13354–13364.CrossRefGoogle ScholarPubMed
Kroehne, V., Heschel, I., Schügner, F., Lasrich, D., Bartsch, J.W. & Jockusch, H. (2008). Use of a novel collegen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts. J Cell Mol Med 12(5a), 1640–1648.CrossRefGoogle Scholar
Leavesley, D.I., Schwartz, M.A., Rosenfeld, M. & Cheresh, D.A. (1993). Integrin beta 1- and beta 3-mediated endothelial cell migration is triggered through distinct signaling mechanisms. J Cell Biol 121(1), 163–170.CrossRefGoogle ScholarPubMed
Leray, A. & Mertz, J. (2006). Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging. Opt Express 14(22), 10565–10573.CrossRefGoogle ScholarPubMed
Lien, S.M., Ko, L.Y. & Huang, T.J. (2009). Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering. Acta Biomater 5(2), 670–679.CrossRefGoogle ScholarPubMed
Lin, P.W., Wu, C.C., Chen, C.H., Ho, H.O., Chen, Y.C. & Sheu, M.T. (2005). Characterization of cortical neuron outgrowth in two- and three-dimensional culture systems. J Biomed Mater Res B Appl Biomater 75(1), 146–157.CrossRefGoogle ScholarPubMed
Ma, P.X. & Elisseeff, J.H. (2005). Scaffolding in Tissue Engineering. Boca Raton, FL: Taylor & Francis.CrossRefGoogle ScholarPubMed
Mohler, W., Millard, A.C. & Campagnola, P.J. (2003). Second harmonic generation imaging of endogenous structural proteins. Methods 29(1), 97–109.CrossRefGoogle ScholarPubMed
Moissoglu, K., Slepchenko, B.M., Meller, N., Horwitz, A.F. & Schwartz, M.A. (2006). In vivo dynamics of Rac-membrane interactions. Mol Biol Cell 17(6), 2770–2779.CrossRefGoogle ScholarPubMed
Mycek, M.-A. & Pogue, B.W. (2003). Handbook of Biomedical Fluorescence. New York: Marcel Dekker.CrossRefGoogle Scholar
Niesner, R., Andresen, V., Neumann, J., Spiecker, H. & Gunzer, M. (2007). The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging. Biophys J 93(7), 2519–2529.CrossRefGoogle ScholarPubMed
Patterson, G., Day, R.N. & Piston, D. (2001). Fluorescent protein spectra. J Cell Sci 114(Pt 5), 837–838.CrossRefGoogle ScholarPubMed
Patterson, G.H. & Piston, D.W. (2000). Photobleaching in two-photon excitation microscopy. Biophys J 78(4), 2159–2162.CrossRefGoogle ScholarPubMed
Ratanavaraporn, J.E.A. (2006). Comparison of gelatin and collagen scaffolds for fibroblast cell culture. J Met Mater Min 16(1), 31–36.Google Scholar
Roeder, B.A., Kokini, K., Sturgis, J.E., Robinson, J.P. & Voytik-Harbin, S.L. (2002). Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure. J Biomech Eng 124(2), 214–222.CrossRefGoogle ScholarPubMed
Sako, Y., Sekihata, A., Yanagisawa, Y., Yamamoto, M., Shimada, Y., Ozaki, K. & Kusumi, A. (1997). Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1. J Microsc 185(Pt 1), 9–20.CrossRefGoogle ScholarPubMed
Seppa, H., Grotendorst, G., Seppa, S., Schiffmann, E. & Martin, G.R. (1982). Platelet-derived growth factor in chemotactic for fibroblasts. J Cell Biol 92(2), 584–588.CrossRefGoogle ScholarPubMed
Seppa, L. (1988). Effects of a sodium fluoride solution and a varnish with different fluoride concentrations on enamel remineralization in vitro . Scand J Dent Res 96(4), 304–309.Google Scholar
Soeller, C. & Cannell, M.B. (1999). Two-photon microscopy: Imaging in scattering samples and three-dimensionally resolved flash photolysis. Microsc Res Techniq 47(3), 182–195.3.0.CO;2-4>CrossRefGoogle ScholarPubMed
Tsien, R.Y. (1998). The green fluorescent protein. Annu Rev Biochem 67, 509–544.CrossRefGoogle ScholarPubMed
Wokosin, D.L., Amos, W.B. & White, J.G. (1998). Detection sensitivity enhancements for fluorescence imaging with multi-photon excitation microscopy. In Engineering in Medicine and Biology Society, 1998. Proceedings of the 20th Annual International Conference of the IEEE, vol. 1704, pp. 1707–1714.Google Scholar
Wolf, K. & Friedl, P. (2009). Mapping proteolytic cancer cell-extracellular matrix interfaces. Clin Exp Metastasis 26(4), 289–298.CrossRefGoogle ScholarPubMed
Wu, R.F., Xu, Y.C., Ma, Z., Nwariaku, F.E., Sarosi, G.A. Jr. & Terada, L.S. (2005). Subcellular targeting of oxidants during endothelial cell migration. J Cell Biol 171(5), 893–904.CrossRefGoogle ScholarPubMed
Zipfel, W.R., Williams, R.M., Christie, R., Nikitin, A.Y., Hyman, B.T. & Webb, W.W. (2003). Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci USA 100(12), 7075–7080.CrossRefGoogle ScholarPubMed