Abstract
Modern imaging approaches are proving important for addressing contemporary issues in the immune system. These approaches are particularly useful for characterizing the complex orchestration of immune responses in vivo. Multicolor, two-photon imaging has been proven to be especially enabling for such studies because of its superior tissue penetration, reduced image degradation by light scattering leading to better resolution and its high image quality deep inside tissues. Here, we examine the functional requirements of two-photon imaging instruments necessary for such immune studies. These requirements include frame rate, spatial resolution and the number of emission channels. We use this discussion as a starting point to compare commercial systems and to introduce a custom technology platform that meets these requirements. This platform is noteworthy because it is very cost-effective, flexible and experimentally useful. Representative data collected with this instrument is used to demonstrate the utility of this platform. Finally, as the field is rapidly evolving, consideration is given to some of the cutting-edge developments in multiphoton microscopy that will likely improve signal strength, depth penetration and/or the experimental usefulness of this approach.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bajenoff M, Egen JG, Koo LY, Laugier JP, Brau F, Glaichenhaus N, Germain RN (2006) Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25:989–1001
Beuneu H, Garcia Z, Bousso P (2006) Cutting edge: cognate CD4 help promotes recruitment of antigen-specific CD8 T cells around dendritic cells. J Immunol 177:1406–1410
Bousso P, Robey E (2003) Dynamics of CD8(+) T cell priming by dendritic cells in intact lymph nodes. Nat Immunol 4:579–585
Bunnell SC, Kapoor V, Trible RP, Zhang W, Samelson LE (2001) Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity 14:315–329
Cahalan M, Parker I (2008) Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs. Annu Rev Immunol 26:585–626
Cahalan MD, Parker I, Wei SH, Miller MJ (2002) Two-photon tissue imaging: seeing the immune system in a fresh light. Nat Rev Immunol 2:872–880
Callamaras N, Parker I (1999) Construction of a confocal microscope for real-time x-y and x-z imaging. Cell Calc 26:271–279
Castellino F, Huang AY, Altan-Bonnet G, Stoll S, Scheinecker C, Germain RN (2006) Chemokines enhance immunity by guiding naive CD8 + T cells to sites of CD4 + T cell-dendritic cell interaction. Nature 440:890–895
Chu K, Lim D, Mertz J (2007) Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination. Opt Lett 32:2846–2848
Codling E, Hill N (2005) Sampling rate effects on measurements of correlated and biased random walks. J Theor Biol 233:573–588
Codling E, Plank M, Benhamou S (2008) Random walk models in biology. J R Soc Interf 5:813–834
Coello Y, Xu B, Miller T, Lozovoy V, Dantus M (2007) Group-velocity dispersion measurements of water, seawater, and ocular components using multiphoton intrapulse interference phase scan. Appl Opt 46:8394–8401
Collier T, Arifler D, Malpica A, Follen M, Richards-Kortum R (2003) Determination of Epithelial Tissue Scattering Coefficient Using Confocal Microscopy. IEEE J Sel Top Quant Electr 9:307–313
Dela Cruz J, Pastirk I, Comstock M, Lozovoy V, Dantus M (2004) Use of coherent control methods through scattering biological tissue to achieve functional imaging. Proc Natl Acad Sci USA 101:16996–17001
Delon J, Bercovici N, Liblau R, Trautmann A (1998) Imaging antigen recognition by naive CD4 + T cells: compulsory cytoskeletal alterations for the triggering of an intracellular calcium response. Eur J Immunol 28:716–729
Delon J, Bercovici N, Raposo G, Liblau R, Trautmann A (1998) Antigen-dependent and -independent Ca2 + responses triggered in T cells by dendritic cells compared with B cells. J Exp Med 188:1473–1484
Delon J, Gregoire C, Malissen B, Darche S, Lemaltre F, Kourilsky P, Abastado JP, Trautmann A (1998) CD8 expression allows T cell signaling by monomeric peptide-MHC complexes. Immunity 9:467–473
Donnert G, Eggeling C, Hell S (2007) Major signal increase in fluorescence microscopy through dark-state relaxation. Nat Methods 4:81–86
Duemani Reddy G, Kelleher K, Fink R, Saggau P (2008) Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat Neurosci 11:713–720
Dustin ML, Bromley SK, Kan Z, Peterson DA, Unanue ER (1997) Antigen receptor engagement delivers a stop signal to migrating T lymphocytes. Proc Natl Acad Sci USA 94:3909–3913
Egeblad M, Ewald A, Askautrud H, Truitt M, Welm B, Bainbridge E, Peeters G, Krummel M, Werb Z (2008) Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Dis Models Mech 1:155–167
Fan G, Fujisaki H, Miyawaki A, Tsay R, Tsien R, Ellisman M (1999) Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys J 76:2412–2420
Frangioni J (2003) In vivo near-infrared fluorescence imaging. Curr Opin Chem Biol 7:626–634
Friedman RS, Jacobelli J, Krummel MF (2005) Mechanisms of T cell motility and arrest: deciphering the relationship between intra- and extracellular determinants. Semin Immunol 17:387–399
Gardner J, Devoss J, Friedman R, Wong D, Tan Y, Zhou X, Johannes K, Su M, Chang H, Krummel M, et al (2008) Deletional tolerance mediated by extrathymic Aire-expressing cells. Science 321:843–847
Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940
Hugues S, Fetler L, Bonifaz L, Helft J, Amblard F, Amigorena S (2004) Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity. Nat Immunol 5:1235–1242
Hugues S, Scholer A, Boissonnas A, Nussbaum A, Combadiere C, Amigorena S, Fetler L (2007) Dynamic imaging of chemokine-dependent CD8 + T cell help for CD8 + T cell responses. Nat Immunol 8:921–930
Ji N, Magee J, Betzig E (2008) High-speed, low-photodamage nonlinear imaging using passive pulse splitters. Nat Methods 5:197–202
König K (2000) Multiphoton microscopy in life sciences. J Microsc 200:83–104
Krummel MF, Sjaastad MD, Wülfing C, Davis MM (2000) Differential Assembly of CD3z and CD4 During T cell Activation. Science 289:1349–1352
Leybaert L, de Meyer A, Mabilde C, Sanderson M (2005) A simple and practical method to acquire geometrically correct images with resonant scanning-based line scanning in a custom-built video-rate laser scanning microscope. J Microsc 219:133–140
Lindquist RL, Shakhar G, Dudziak D, Wardemann H, Eisenreich T, Dustin ML, Nussenzweig MC (2004) Visualizing dendritic cell networks in vivo. Nat Immunol 5:1243–1250
Livet J, Weissman T, Kang H, Draft R, Lu J, Bennis R, Sanes J, Lichtman J (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450:56–62
Majewska A, Yiu G, Yuste R (2000) A custom-made two-photon microscope and deconvolution system. Pflugers Arch 441:398–408
McConnell G (2006) Improving the penetration depth in multiphoton excitation laser scanning microscopy. J Biomed Opt 11:054020
Mempel TR, Henrickson SE, von Andrian UH (2004) T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427:154–159
Miller MJ, Safrina O, Parker I, Cahalan MD (2004) Imaging the single cell dynamics of CD4 + T cell activation by dendritic cells in lymph nodes. J Exp Med 200:847–856
Miller MJ, Wei SH, Parker I, Cahalan MD (2002) Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296:1869–1873
Negulescu PA, Krasieva TB, Khan A, Kerschbaum HH, Cahalan MD (1996) Polarity of T cell shape, motility, and sensitivity to antigen. Immunity 4:421–430
Nguyen Q, Tsai P, Kleinfeld D (2006) MPScope: a versatile software suite for multiphoton microscopy. J Neurosci Methods 156:351–359
Nguyen QT, Callamaras N, Hsieh C, Parker I (2001) Construction of a two-photon microscope for video-rate Ca(2+) imaging. Cell Calc 30:383–393
Nikolenko V, Nemet B, Yuste R (2003) A two-photon and second-harmonic microscope. Methods 30:3–15
Okada T, Miller MJ, Parker I, Krummel MF, Neighbors M, Hartley SB, O’Garra A, Cahalan MD, Cyster JG (2005) Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol 3:e150
Othmer H, Dunbar S, Alt W (1988) Models of dispersal in biological systems. J Math Biol 26:263–298
Phan TG, Grigorova I, Okada T, Cyster JG (2007) Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nat Immunol 8:992–1000
Poenie M, Tsien RY, Schmitt-Verhulst AM (1987) Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets. EMBO J 6:2223–2232
Pologruto T, Sabatini B, Svoboda K (2003) ScanImage: flexible software for operating laser scanning microscopes. Biomed Eng Online 2:13
Qi H, Egen J, Huang A, Germain R (2006) Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312:1672–1676
Reddy G, Saggau P (2005) Fast three-dimensional laser scanning scheme using acousto-optic deflectors. J Biomed Opt 10:064038
Ridsdale A, Micu I, Stys P (2004) Conversion of the Nikon C1 confocal laser-scanning head for multiphoton excitation on an upright microscope. Appl Opt 43:1669–1675
Sabatos CA, Doh J, Chakravarti S, Friedman RS, Prandurangi PG, Tooley AJ, Krummel MF (2008) A synaptic basis for paracrine interleukin-2 signalling during homotypic T cell interaction. Immunity 29:238–248.
Saggau P (2006) New methods and uses for fast optical scanning. Curr Opin Neurobiol 16:543–550
Sanderson MJ, Parker I (2003) Video-rate confocal microscopy. Methods Enzymol 360:447–481
Sanderson MJ (2004) Acquisition and correction of multiple real-time images for laser-scanning microscopy. Microsc Anal 18:17–23
Schaefer BC, Marrack P, Fanger GR, Kappler JW, Johnson GL, Monks CRF (1999) Live cell fluorescence imaging of T cell MEKK2: Redistribution and activation in response to antigen stimulation of the T cell receptor. Immunity 11:411–421
Schelhas L, Shane J, Dantus M (2006) Advantages of ultrashort phase-shaped pulses for selective two-photon activation and biomedical imaging. Nanomedicine 2:177–181
Shakhar G, Lindquist RL, Skokos D, Dudziak D, Huang JH, Nussenzweig MC, Dustin ML (2005) Stable T cell-dendritic cell interactions precede the development of both tolerance and immunity in vivo. Nat Immunol 6:707–714
Stoll S, Delon J, Brotz TM, Germain RN (2002) Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science 296:1873–1876
Tang Q, Adams JY, Tooley AJ, Bi M, Fife BT, Serra P, Santamaria P, Locksley RM, Krummel MF, Bluestone JA (2006) Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat Immunol 7:83–92
Theer P, Hasan M, Denk W (2003) Two-photon imaging to a depth of 1000 microm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt Lett 28:1022–1024
Varma R, Campi G, Yokosuka T, Saito T, Dustin ML (2006) T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25:117–127
Williams R, Zipfel W, Webb W (2001) Multiphoton microscopy in biological research. Curr Opin Chem Biol 5:603–608
Wülfing C, Rabinowitz JD, Beeson C, Sjaastad MD, McConnell HM, Davis MM (1997) Kinetics and extent of T cell activation as measured with the calcium signal. J Exp Med 185:1815–1825
Yokosuka T, Sakata-Sogawa K, Kobayashi W, Hiroshima M, Hashimoto-Tane A, Tokunaga M, Dustin ML, Saito T (2005) Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat Immunol 6:117–127
Zhu G, van Howe J, Durst M, Zipfel W, Xu C (2005) Simultaneous spatial and temporal focusing of femtosecond pulses. Opt Exp 13:2153–2159
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Bullen, A., Friedman, R.S., Krummel, M.F. (2009). Two-Photon Imaging of the Immune System: A Custom Technology Platform for High-Speed, Multicolor Tissue Imaging of Immune Responses. In: Dustin, M., McGavern, D. (eds) Visualizing Immunity. Current Topics in Microbiology and Immunology, vol 334. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-93864-4_1
Download citation
DOI: https://doi.org/10.1007/978-3-540-93864-4_1
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-540-93862-0
Online ISBN: 978-3-540-93864-4
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)