Abstract
Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment—the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Tang JY, van Panhuys N, Kastenmuller W, Germain RN (2013) The future of immunoimaging - deeper, bigger, more precise, and definitively more colorful. Eur J Immunol 43:1407–1412
Schubert W, Bonnenkoh B, Pommer AJ et al (2006) Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nat Biotechnol 24(10):1270–1278. https://doi.org/10.1038/nbt1250
Holzwarth K, Köhler R, Philipsen L et al (2018) Multiplexed fluorescence microscopy reveals heterogeneity among stromal cells in mouse bone marrow sections. Cytometry A 93(9):876–888. https://doi.org/10.1002/cyto.a.23526
Giesen C, Wang HA, Schapiro D et al (2014) Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods 11(4):417–422. https://doi.org/10.1038/nmeth.2869
Rakhymzhan A, Leben R, Zimmermann H et al (2017) Synergistic strategy for multicolor two-photon microscopy: application to the analysis of germinal center reactions in vivo. Sci Rep 7(1):7101. https://doi.org/10.1038/s41598-017-07165-0
Hauser AE, Junt T, Mempel TR et al (2007) Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns. Immunity 26(5):655–667
Shcherbakova DM, Verkhusha VV (2013) Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat Methods 10(8):751–754. https://doi.org/10.1038/nmeth.2521
Shcherbo D, Shemiakina II, Ryabova AV et al (2010) Near-infrared fluorescent proteins. Nat Methods 7(10):827–829. https://doi.org/10.1038/nmeth.1501
Entenberg D, Wyckoff J, Glicorijevic B et al (2011) Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging. Nat Protoc 6:1500–1520. https://doi.org/10.1038/nprot.2011.376
Ricard C, Debarbieux FC (2014) Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment. Front Cell Neurosci 8:57. https://doi.org/10.3389/fncel.2014.00057
Herz J, Siffrin V, Hauser AE et al (2010) Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator. Biophys J 98:715–723. https://doi.org/10.1016/j.bpj.2009.10.035
Mahou P, Zimmerley M, Loulier K et al (2012) Multicolor two-photon tissue imaging by wavelength mixing. Nat Methods 9:815–818. https://doi.org/10.1038/nmeth.2098
Tu H, Boppart SA (2013) Coherent fiber supercontinuum for biophotonics. Laser Photon Rev 7(5):628–645. https://doi.org/10.1002/lpor.201200014
Zimmermann T (2005) Spectral imaging and linear unmixing in light microscopy. Adv Biochem Eng Biotechnol 95:245–265
Snippert HJ, van der Flier LG, Sato T et al (2010) Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143:134–144. https://doi.org/10.1016/j.cell.2010.09.016
Seibler J, Zevnik B, Küter-Luks B et al (2003) Rapid generation of inducible mouse mutants. Nucleic Acids Res 31(4):e12
Ulbricht C, Lindquist RL, Tech L, Hauser AE (2017) Tracking plasma cell differentiation in living mice with two-photon microscopy. Methods Mol Biol 1623:37–50. https://doi.org/10.1007/978-1-4939-7095-7_3
Niesner R, Roth W, Gericke KH (2004) Photophysical aspects of single-molecule detection by two-photon excitation with consideration of pulsed illumination. ChemPhysChem 5(5):678–687
Niesner R, Gericke KH (2006) Quantitative determination of the single molecule detection regime in fluorescence fluctuation microscopy by means of photon counting histogram analysis. J Chem Phys 124(13):134704
Acknowledgments
We thank Robert Günther and Peggy Mex for excellent technical support. Financial support from the German Research Council (DFG) under grant TRR130 (C01 to R.N., C01, P17 to A.E.H. and P11, C03 to T.H.W.), FOR2165/2 (NI1167/4-2 to R.N. and HA5354/6-2 to A.E.H.), and HA5354/8-1 (SPP1937) to A.E.H. is greatly acknowledged.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rakhymzhan, A., Acs, A., Leben, R., Winkler, T.H., Hauser, A.E., Niesner, R.A. (2021). Method for Multiplexed Dynamic Intravital Multiphoton Imaging. In: Zamir, E. (eds) Multiplexed Imaging. Methods in Molecular Biology, vol 2350. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1593-5_10
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1593-5_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1592-8
Online ISBN: 978-1-0716-1593-5
eBook Packages: Springer Protocols