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  • Review Article
  • Published:

Dynamic imaging of the immune system: progress, pitfalls and promise

Nature Reviews Immunology volume 6, pages 497–507 (2006)Cite this article

Key Points

  • This Review provides an overview of the technical and experimental differences between single- and two-photon imaging. The different depths of imaging in complex tissues that can be achieved using the two methods and the benefits of the longer wavelength laser illumination used in two-photon imaging in avoiding phototoxicity are emphasized.

  • A detailed description of methods for achieving fluorescent labelling of cells for imaging, and a comparison of organic dyes, intrinsic fluorescent protein expression and quantum dots is provided.

  • The advantages and disadvantages of using tissue explants and intravital methods are discussed, with an emphasis on how oxygenation and perfusion affect cell mobility in explants.

  • Methods for maintaining the physiological state of imaged tissues and measuring how well such a state has been preserved are discussed.

  • The article describes how imaging data are collected and analysed, along with the pitfalls to be aware of in collecting, processing, and evaluating such data.

  • Discussion of some of the crucial aspects of microscope design that affect the collection of useful imaging data involving rapidly moving cells is included in the manuscript.

  • This Review provides a guide to existing studies using dynamic-imaging methods in immunological research and a summary of ongoing efforts to extend the technology to currently inaccessible tissues, to the molecular level and to the functional behaviour of cells.

Abstract

Both innate and adaptive immunity are dependent on the migratory capacity of myeloid and lymphoid cells. Effector cells of the innate immune system rapidly enter infected tissues, whereas sentinel dendritic cells in these sites mobilize and transit to lymph nodes. In these and other secondary lymphoid tissues, interactions among various cell types promote adaptive humoral and cell-mediated immune responses. Recent advances in light microscopy have allowed direct visualization of these events in living animals and tissue explants, which allows a new appreciation of the dynamics of immune-cell behaviour. In this article, we review the basic techniques and the tools used for in situ imaging, as well as the limitations and potential artefacts of these methods.

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Figure 1: Detection of four fluorescent signals with three photomultiplier tubes.

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Acknowledgements

The authors wish to thank the Howard Hughes Medical Institute for their sponsorship of the conference during which many of the issues covered in this Review were discussed. They also gratefully acknowledge the free exchange of data and ideas (many unpublished) among investigators active in the imaging field during that meeting, information that was essential to the formulation of this article. Thanks also to J. Egen for his thoughtful comments on drafts of this manuscript. The authors' laboratories have been supported by funds from the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, the National Institutes of Health (NIH),USA (R.N.G.), the Howard Hughes Medical Institute (M.C.N.) and grants from the NIH (M.L.D. and M.C.N.).

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Authors and Affiliations

  1. Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Ronald N. Germain

  2. Department of Pathology and Immunology, Washington University School of Medicine, St Louis, 63110–1093, Missouri, USA

    Mark J. Miller

  3. Skirball Institute of Biomolecular Medicine and Department of Pathology, Program in Molecular Pathogenesis, New York University School of Medicine, New York, 10016, New York, USA

    Michael L. Dustin

  4. Laboratory of Molecular Immunology and the Howard Hughes Medical Institute, The Rockefeller University, New York, 10021, New York, USA

    Michel C. Nussenzweig

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  1. Ronald N. Germain
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  4. Michel C. Nussenzweig
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Correspondence to Ronald N. Germain.

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Glossary

Confocal microscopy

A form of fluorescence microscopy in which out-of-focus signals are rejected by an aperture that restricts all light from reaching the detector except that originating from the focal plane of the excitation spot.

Two-photon microscopy

A fluorescence-imaging technique that takes advantage of the fact that fluorescent molecules can absorb two photons nearly simultaneously during excitation before they emit light. This technique allows all emitted photons to contribute to a useful image.

Positron emission tomography

An imaging method that depends on the three-dimensional detection of (positrons) radiation from a probe that is typically localized to a cell by direct ex vivo labelling or in situ metabolic conversion of a precursor compound.

Magnetic resonance imaging

A method that uses detection of changes in the alignment of protons in a strong magnetic field when they are perturbed by radio wave pulses to generate structural information about an object in that magnetic field.

Luminescence imaging

A technique that uses photons emitted by the process of luminescence, rather than fluorescence, to obtain an image of cells in a living animal. This method is extremely sensitive and non-invasive but generates data of much lower resolution than microscope-based fluorescent imaging.

Knock-in technology

The introduction of a transgene into a precise location in the genome, rather than a random integration site. Knocking-in uses the same technique of homologous recombination as a knockout strategy but the targeting vector is designed to allow expression of the introduced transgene under control of the regulatory elements of the targeted gene.

BAC transgenic technology

A method for creating genetically altered mice in which very large segments of mouse genomic DNA are propagated in bacteria and used to achieve physiological patterns of gene expression. This technique avoids the need to create knock-in mice by homologous recombination in embryonic stem cells.

SIN vectors

Retroviral or lentiviral vectors that contain mutations that inactivate the enhancer element in the 3′ LTR (long terminal repeat). Because the sequence of the 3′ LTR is used to reconstitute the 5′ LTR during reverse transcription, these vectors 'self-inactivate' the 5′ LTR enhancer before integration into the host-cell DNA. This allows exogenous gene regulatory sequences downstream of the 5′ LTR to control gene expression after integration.

Emission spectrum

A quantitative representation of the wavelengths (energies) of the photons emitted from a fluorescent compound after it is excited by shorter wavelength (more energetic) photons from an illumination source.

Excitation optimum

The wavelength of incident light that is best absorbed by and causes maximal emission from a fluorescent compound.

Quantum dot

A nanocrystalline semi-conductor of extremely small size (10–50 nm) that results in its absorption of incident photons, followed by the emission of photons at a slightly longer wavelength. Because of a phenomenon called the quantum confinement effect, the colour (wavelength) of the emitted light is determined by the size of the nanocrystal.

Water-dipping lens

An objective lens for a microscope that is optimized for use with its front surface in contact with an aqueous solution, because there is an improved match in refractive index between the glass and buffer solution that limits spherical aberration in the image.

Second harmonic emission

The non-radiative production of frequency-doubled polarized light emission from a highly ordered (anisotropic) material on illumination by a laser beam. In practical terms, the production of polarized light emission from extracellular matrix materials such as collagen when subjected to two-photon illumination in the absence of fluorochrome labelling.

Galvanometers

A device that uses electric currents to control the mechanical displacement of an object such as a scan mirror that in turn directs a laser beam.

Heisenberg's Uncertainty Principle

The concept that measurement of the properties of an object, in particular momentum and position, cannot be accomplished with complete accuracy. Sometimes used (with some license) to encompass the 'observer effect', which indicates that the mere attempt to measure such properties changes them from their intrinsic state.

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Germain, R., Miller, M., Dustin, M. et al. Dynamic imaging of the immune system: progress, pitfalls and promise. Nat Rev Immunol 6, 497–507 (2006). https://doi.org/10.1038/nri1884

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