Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Two-photon tissue imaging: seeing the immune system in a fresh light

Key Points

  • Two main techniques have been used to analyse the immune system. In vivo experiments examine cell populations in their natural environment, but they cannot provide information about individual cells. In vitro experiments determine subcellular and molecular details, but they cannot replicate adequately the physiological environment. Therefore, new techniques, involving fluorescent labels, are required to analyse single cells in intact tissues in real time.

  • Three main problems limit optical resolution when attempting to visualize fluorescently labelled cells in living tissues. First, the high-numerical-aperture lenses that are required have a narrow depth of field, so that fluorescent label from above and below the focus obscures the image. Second, biological tissues scatter light, which reduces image contrast. Third, the intense excitation light causes photodamage and photobleaching.

  • A partial solution is provided by confocal microscopy, which provides a sharp 'optical section' at a given depth by rejecting out-of-focus fluorescence from above or below the focal plane. But, light scattering limits the depth of imaging and the specimen is susceptible to photodamage.

  • Two-photon microscopy has the advantages of greater imaging depth (owing to reduced light scattering of the long wavelengths that are used for excitation) and minimal photobleaching (as excitation is confined to the focal plane).

  • Two-photon excitation involves the simultaneous absorption by a fluorophore of energy from two photons, each of which contributes one half of the total energy required to induce fluorescence. The probability of two-photon excitation falls off rapidly from the focal point.

  • Pulsed lasers (such as the titanium-sapphire laser) are required to provide the necessary high photon density at the focal spot without vaporizing the specimen. The peak power during a pulse is extremely high, but the average laser power is relatively low.

  • The choice of objective lens is also important to maximize the numerical aperture while maintaining imaging depth. 'Dipping' water-immersion objectives provide the best compromise.

  • Two-photon microscopy allows imaging in as many as six dimensions (three spatial dimensions, time, intensity and wavelength of more than one probe). This creates problems in terms of storage, analysis and representation of the data that will require new solutions, such as the use of virtual-reality technology.

  • As an example, two-photon techniques have been used to examine the movement of T cells in lymph nodes and their response to antigen. These studies have indicated that both the single-encounter model and the serial-encounter model of antigen presentation might be relevant physiologically.

Abstract

Many lymphocyte functions, such as antigen recognition, take place deep in densely populated lymphoid organs. Because direct in vivo observation was not possible, the dynamics of immune-cell interactions have been inferred or extrapolated from in vitro studies. Two-photon fluorescence excitation uses extremely brief (<1 picosecond) and intense pulses of light to 'see' directly into living tissues, to a greater depth and with less phototoxicity than conventional imaging methods. Two-photon microscopy, in combination with newly developed indicator molecules, promises to extend single-cell approaches to the in vivo setting and to reveal in detail the cellular collaborations that underlie the immune response.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: In vitro versus in vivo approaches to immunology.
Figure 2: Principles of confocal and two-photon microscopy.
Figure 3: Two-photon excitation of fluorophors by spatial and temporal compression of photons.
Figure 4: Multi-dimensional two-photon microscopy: tissue imaging and tracking cell proliferation.

Similar content being viewed by others

References

  1. Donnadieu, E., Bismuth, G. & Trautmann, A. Antigen recognition by helper T cells elicits a sequence of distinct changes of their shape and intracellular calcium. Curr. Biol. 4, 584–595 (1994).

    Article  CAS  PubMed  Google Scholar 

  2. Negulescu, P. A., Shastri, N. & Cahalan, M. D. Intracellular calcium dependence of gene expression in single T lymphocytes. Proc. Natl Acad. Sci. USA 91, 2873–2877 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fanger, C. M., Hoth, M., Crabtree, G. R. & Lewis, R. S. Characterization of T-cell mutants with defects in capacitative calcium entry: genetic evidence for the physiological roles of CRAC channels. J. Cell. Biol. 131, 655–667 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Negulescu, P. A., Krasieva, T. B., Khan, A., Kerschbaum, H. H. & Cahalan, M. D. Polarity of T-cell shape, motility and sensitivity to antigen. Immunity 4, 421–430 (1996).This study describes the T cell as a motile, polarized antigen sensor, for which Ca2+ functions as a stop signal.

    Article  CAS  PubMed  Google Scholar 

  5. Delon, J., Bercovici, N., Raposo, G., Liblau, R. & Trautmann, A. Antigen-dependent and -independent Ca2+ responses triggered in T cells by dendritic cells compared with B cells. J. Exp. Med. 188, 1473–1484 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dolmetsch, R. E., Xu, K. & Lewis, R. S. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392, 933–936 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Wei, X., Tromberg, B. J. & Cahalan, M. D. Mapping the sensitivity of T cells with an optical trap: polarity and minimal number of receptors for Ca2+ signaling. Proc. Natl Acad. Sci. USA 96, 8471–8476 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dustin, M. L., Bromley, S. K., Kan, Z., Peterson, D. A. & Unanue, E. R. Antigen-receptor engagement delivers a stop signal to migrating T lymphocytes. Proc. Natl Acad. Sci. USA 94, 3909–3913 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. van der Merwe, P. A., Davis, S. J., Shaw, A. S. & Dustin, M. L. Cytoskeletal polarization and redistribution of cell-surface molecules during T-cell antigen recognition. Semin. Immunol. 12, 5–21 (2000).

    Article  CAS  Google Scholar 

  10. Bromley, S. K. et al. The immunological synapse. Annu. Rev. Immunol. 19, 375–396 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Kupfer, A., Singer, S. J., Janeway, C. A. Jr & Swain, S. L. Coclustering of CD4 (L3T4) molecule with the T-cell receptor is induced by specific direct interaction of helper T cells and antigen-presenting cells. Proc. Natl Acad. Sci. USA 84, 5888–5892 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. McConnell, H. M., Watts, T. H., Weis, R. M. & Brian, A. A. Supported planar membranes in studies of cell–cell recognition in the immune system. Biochim. Biophys. Acta 864, 95–106 (1986).

    Article  CAS  PubMed  Google Scholar 

  13. Watts, T. H., Gaub, H. E. & McConnell, H. M. T-cell-mediated association of peptide antigen and major histocompatibility complex protein detected by energy transfer in an evanescent wave-field. Nature 320, 179–181 (1986).

    Article  CAS  PubMed  Google Scholar 

  14. Dustin, M. L. & Springer, T. A. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341, 619–624 (1989).

    CAS  PubMed  Google Scholar 

  15. Dustin, M. L. & Springer, T. A. Role of lymphocyte adhesion receptors in transient interactions and cell locomotion. Annu. Rev. Immunol. 9, 27–66 (1991).

    Article  CAS  PubMed  Google Scholar 

  16. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T-cell activation. Science 285, 221–227 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Zal, T., Zal, M. A. & Gascoigne, N. R. Inhibition of T-cell-receptor–coreceptor interactions by antagonist ligands visualized by live FRET imaging of the T-hybridoma immunological synapse. Immunity 16, 521–534 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Dustin, M. L. & de Fougerolles, A. R. Reprogramming T cells: the role of extracellular matrix in coordination of T-cell activation and migration. Curr. Opin. Immunol. 13, 286–290 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Lanzavecchia, A. & Sallusto, F. The instructive role of dendritic cells on T-cell responses: lineages, plasticity and kinetics. Curr. Opin. Immunol. 13, 291–298 (2001).This paper questions the requirement for stable interactions between T cells and antigen-presenting cells.

    Article  CAS  PubMed  Google Scholar 

  20. Gunzer, M. et al. Antigen presentation in extracellular matrix: interactions of T cells with dendritic cells are dynamic, short lived and sequential. Immunity 13, 323–332 (2000).

    Article  CAS  PubMed  Google Scholar 

  21. Dustin, M. L., Allen, P. M. & Shaw, A. S. Environmental control of immunological synapse formation and duration. Trends Immunol. 22, 192–194 (2001).This article reviews the immunological synapse as a stable organizing element and proposes that the environment might influence the stability of the synapse.

    Article  CAS  PubMed  Google Scholar 

  22. Friedl, P. & Gunzer, M. Interaction of T cells with APCs: the serial encounter model. Trends Immunol. 22, 187–191 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Jung, T., Schauer, U., Heusser, C., Neumann, C. & Rieger, C. Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159, 197–207 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. Prussin, C. & Metcalfe, D. D. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J. Immunol. Methods 188, 117–128 (1995).

    Article  CAS  PubMed  Google Scholar 

  25. Altman, J. D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. Lyons, A. B., Hasbold, J. & Hodgkin, P. D. Flow cytometric analysis of cell-division history using dilution of carboxyfluorescein diacetate succinimidyl ester, a stably integrated fluorescent probe. Methods Cell Biol. 63, 375–398 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Ingulli, E., Mondino, A., Khoruts, A. & Jenkins, M. K. In vivo detection of dendritic-cell antigen presentation to CD4+ T cells. J. Exp. Med. 185, 2133–2141 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Okada, T. et al. Chemokine requirements for B-cell entry to lymph nodes and Peyer's patches. J. Exp. Med. 196, 65–75 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cyster, J. G. Chemokines and cell migration in secondary lymphoid organs. Science 286, 2098–2102 (1999).

    Article  CAS  PubMed  Google Scholar 

  30. Kawakami, N. et al. Green fluorescent protein-transgenic mice: immune functions and their application to studies of lymphocyte development. Immunol. Lett. 70, 165–171 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Schleicher, U., Rollinghoff, M. & Gessner, A. A stable marker for specific T cells: a TCRα/green fluorescent protein (GFP) fusion protein reconstitutes a functionally active TCR complex. J. Immunol. Methods 246, 165–174 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Naramura, M., Hu, R. J. & Gu, H. Mice with a fluorescent marker for interleukin-2 gene activation. Immunity 9, 209–216 (1998).

    Article  CAS  PubMed  Google Scholar 

  33. Hu-Li, J. et al. Regulation of expression of IL-4 alleles: analysis using a chimeric GFP/IL-4 gene. Immunity 14, 1–11 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. von Andrian, U. H. Immunology. T-cell activation in six dimensions. Science 296, 1815–1817 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Becker, M. D. et al. Intraocular in vivo imaging of activated T lymphocytes expressing green-fluorescent protein after stimulation with endotoxin. Graefes Arch. Clin. Exp. Ophthalmol. 239, 609–612 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Kedl, R. M. et al. T cells compete for access to antigen-bearing antigen-presenting cells. J. Exp. Med. 192, 1105–1113 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gretz, J. E., Anderson, A. O. & Shaw, S. Cords, channels, corridors and conduits: critical architectural elements facilitating cell interactions in the lymph-node cortex. Immunol. Rev. 156, 11–24 (1997).

    Article  CAS  PubMed  Google Scholar 

  38. Gretz, J. E., Norbury, C. C., Anderson, A. O., Proudfoot, A. E. & Shaw, S. Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex. J. Exp. Med. 192, 1425–1440 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wulfing, C., Sjaastad, M. D. & Davis, M. M. Visualizing the dynamics of T-cell activation: intracellular adhesion molecule 1 migrates rapidly to the T-cell/B-cell interface and acts to sustain calcium levels. Proc. Natl Acad. Sci. USA 95, 6302–6307 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Schaefer, B. C. et al. 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 (1999).

    Article  CAS  PubMed  Google Scholar 

  41. Krummel, M. F., Sjaastad, M. D., Wulfing, C. & Davis, M. M. Differential clustering of CD4 and CD3ξ during T-cell recognition. Science 289, 1349–1352 (2000).

    Article  CAS  PubMed  Google Scholar 

  42. Reichert, P., Reinhardt, R. L., Ingulli, E. & Jenkins, M. K. Cutting edge: in vivo identification of TCR redistribution and polarized IL-2 production by naive CD4 T cells. J. Immunol. 166, 4278–4281 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Ojcius, D. M., Niedergang, F., Subtil, A., Hellio, R. & Dautry-Varsat, A. Immunology and the confocal microscope. Res. Immunol. 147, 175–188 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Mayer, M. G. Elementary processes with two-quantum transitions. Ann. d. Physik 9, 273–294 (1931).

    Article  Google Scholar 

  45. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  CAS  PubMed  Google Scholar 

  46. Patterson, G. H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).

    Article  CAS  PubMed  Google Scholar 

  47. Jenkins, M. K. et al. In vivo activation of antigen-specific CD4 T cells. Annu. Rev. Immunol. 19, 23–45 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Miller, M. J., Wei, S. H., Parker, I. & Cahalan, M. D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).The first use of two-photon microscopy to observe the dynamics of T- and B-cell motility in an intact lymphoid organ; T cells were observed with and without antigen challenge.

    Article  CAS  PubMed  Google Scholar 

  49. Bousso, P., Bhakta, N. R., Lewis, R. S. & Robey, E. Dynamics of thymocyte–stromal cell interactions visualized by two-photon microscopy. Science 296, 1876–1880 (2002).Two-photon microscopy was used to image interactions between thymocytes and stromal cells in a reaggregated thymic organ culture system during positive selection.

    Article  CAS  PubMed  Google Scholar 

  50. Wei, S. H., Miller, M. J., Cahalan, M. D. & Parker, I. Two-photon imaging in intact lymphoid tissue. Adv. Exp. Med. Biol. (in the press).

  51. Stoll, S., Delon, J., Brotz, T. M. & Germain, R. N. Dynamic imaging of T-cell–dendritic cell interactions in lymph nodes. Science 296, 1873–1876 (2002).Confocal imaging of T cells interacting with dendritic cells in intact lymph nodes.

    Article  PubMed  Google Scholar 

  52. Caldwell, C. C. et al. Differential effects of physiologically relevant hypoxic conditions on T-lymphocyte development and effector functions. J. Immunol. 167, 6140–6149 (2001).

    Article  CAS  PubMed  Google Scholar 

  53. Ley, K., Pries, A. R. & Gaehtgens, P. A versatile intravital microscope design. Int. J. Microcirc. Clin. Exp. 6, 161–167 (1987).

    CAS  PubMed  Google Scholar 

  54. Dunne, J. L., Ballantyne, C. M., Beaudet, A. L. & Ley, K. Control of leukocyte rolling velocity in TNF-α-induced inflammation by LFA-1 and Mac-1. Blood 99, 336–341 (2002).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank J. Cyster and T. Okada for providing the immunohistochemistry picture in Fig. 1b, and S. Schoenberger and M. van Stipdonk for their permission to show Fig. 4b. M.D.C. and I.P. are supported by grants from the National Institutes of Health.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Michael D. Cahalan.

Related links

Related links

DATABASES

LocusLink

CD3ζ

CD4

IL-2

IL-4

NFAT

Swiss-Prot

BFP

CFP

GFP

YFP

FURTHER INFORMATION

Center for Ultrastructural Research, University of Georgia

Handbook of Fluorescent Probes and Research Products

Ian Parker's lab

Leiden Institue of Physics: Physics of Life Processes

Michael Cahalan's lab

Multiphoton Microscopy

Universal Imaging Corporation™ (MetaMorph®)

Glossary

FLUORESCENCE RESONANCE ENERGY TRANSFER

(FRET). This is used to measure protein–protein interactions microscopically or by a FACS-based method. Proteins fused to cyan, yellow or red fluorescent proteins are expressed and their interactions assessed by measuring the energy transfer between fluors, which can occur only if the proteins interact physically.

BRIGHT-FIELD MICROSCOPY

The original, and most commonly used, form of microscopy, in which the specimen is viewed by transmitted light from a condenser lens.

NOMARSKI OPTICS

One of several implementations of differential interference contrast (DIC) techniques for use with bright-field microscopy. DIC converts optical gradients in a specimen (differences in thickness, slope or refractive index) to differences in intensity, which gives a pseudo three-dimensional appearance to objects that would otherwise appear featureless.

FLUORESCENCE MICROSCOPY

The visualization of fluorescence in a specimen — either natural autofluorescence or after staining with fluorescent probes. Usually, this is achieved using an epi-fluorescence microscope, in which the excitation light is directed by a dichroic mirror through the objective lens, and fluorescence emission is viewed through the same lens after blocking excitation light with a barrier filter.

FURA-2 RATIOMETRIC IMAGING

Calcium imaging using the calcium-sensitive dye fura-2. When excited at short ultra-violet wavelengths (350 nm), the fluorescence of fura-2 increases with increasing calcium concentration, whereas fluorescence decreases with increasing calcium concentration at longer wavelengths (380 nm). By forming a ratio of successive images obtained at each wavelength, it is possible to obtain a measure of absolute free calcium concentration.

DECONVOLUTION MICROSCOPY

The use of software to correct partially for diffraction-limited blurring by a microscope and thereby enhance the resolution of three-dimensional images obtained by wide-field or confocal microscopy.

CONFOCAL MICROSCOPY

A form of fluorescence (or reflected light) microscopy in which out-of-focus signals are rejected by measuring through an aperture that restricts all light except that originating from the focused excitation spot.

LIPID-BILAYER IMAGING

This involves phospholipid with protein constituents deposited in a bilayer on a glass surface; it is used to study cell–cell interactions, such as the immunological synapse.

COLLAGEN GEL MATRIX

A culture system consisting of a three-dimensional collagen environment that mimics a physiological substrate for the study of immune-cell motility and function.

CFSE LABELLING

CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester) is a green-fluorescent dye that binds covalently to intracellular proteins, providing a stable cell label. When a cell divides, the dye is diluted equally in the daughter cells and fluorescence is halved with each generation, which allows up to 7–10 cell divisions to be detected by flow cytometry.

NUMERICAL APERTURE

(NA). The light-gathering ability of a microscope objective. The NA is equal to the refractive index of the medium multiplied by the sine of half the angular aperture.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cahalan, M., Parker, I., Wei, S. et al. Two-photon tissue imaging: seeing the immune system in a fresh light. Nat Rev Immunol 2, 872–880 (2002). https://doi.org/10.1038/nri935

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nri935

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing