Shine a light: Imaging the immune system - Wiley Online Library

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Milka Sarris and Alexander G. Betz

DOI 10.1002/eji.200839026

Eur. J. Immunol. 2009. 39: 1188–1202

Review

Shine a light: Imaging the immune system Milka Sarris and Alexander G. Betz Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK Intra vital microscopy and whole-body imaging promise to revolutionize how we study the immune system. They compel by the intrinsic beauty of the images obtained and the undeniable direct biological relevance of the observations. However, it is important to remember that in many cases, fundamental insights into the underlying biological processes have already been obtained using ex vivo reductionist approaches. Indeed, it is likely that with the advent of microfluidics, new and exciting avenues will open up for ex vivo experimentation. Here, we give a brief but comprehensive overview of the various imaging techniques available, their relative strengths and shortcomings and how these tools have been used to get us to where we are today. The challenge for the future will be to apply the most suitable technology and to integrate the findings across various imaging disciplines to build a unified, comprehensive ‘‘big picture’’ of the immune system.

Key words: Imaging . Microscopy . Technology

During a comparative study of digestive organs, Elie Metchnikoff was intrigued by the fact that certain cells that play no role in digestion, nevertheless have the ability to ingest foreign bodies [1]. The ‘‘model system’’ he used to study this phenomenon was the starfish larva, which is transparent enough to observe individual cells moving within. Metchnikoff observed that when he inserted a small splinter into the larvae, some cells accumulated at the point of insult and tried to ingest the foreign body. He had discovered phagocytosis, a phenomenon he demonstrated to be ubiquitous throughout the Animal Kingdom [2]. As higher organisms were not suited to direct microscopic examination, Metchnikoff proceeded to isolate phagocytic cells from the blood of higher organisms and demonstrated that these cells ingest and destroy microorganisms. Based on his observation, he introduced the concept of cellular immunity. Thus, it is fair to say that microscopic imaging has always been at the center of immunological research from its very conception. Initially however, immunologists had no tools with which to observe the cells involved in immune responses in higher organisms in vivo. They could use either tissue sections to reconstitute what had happened from snapshots or they could study specific aspects of the immune response with isolated cell populations ex vivo. The

Correspondence: Dr. Alexander G. Betz e-mail: [email protected]

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

contributions of tissue imaging in immunology over the past century have been recently reviewed [3]. Here, we provide a broad overview of the multitude of imaging approaches that have been applied in immunology, ranging from ex vivo single molecule detection to in vivo whole-body imaging and microfluidic ‘‘lab on a chip’’ technology for immunological studies.

Ex vivo studies Many of the functions of the immune system can be mimicked ex vivo using primary cell populations. Early separation techniques relied on the physical characteristics of the cells. Velocity sedimentation was used to sort the cells according to size [4] and differential adherence in order to separate subpopulations based on cell–matrix interactions [4]. As more cell surface markers were identified using antisera, specific separation methods were developed. Antibody-labeled cells were either removed by lysis through exposure to complement or enriched by passing through affinity columns [4]. With the introduction of the hybridoma technique [5], monoclonal antibodies directed against specific targets could be generated readily, leading to a rapid increase in the availability of specific markers. This in turn nurtured the development of more advanced cell purification methods. Flow cytometry (Table 1) allows the identification, analysis and bulk sorting of cells based on their optical properties [6].

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Table 1. Cell identification/separation methods Cell identification and separation

Advantages

Disadvantages

More information

Flow cytometry

Both identification and separation

Cells at times exposed to high shear

[6]

of cell populations based on size, granularity and fluorescence

forces during sorting Too slow for bulk cell separations

Various types of analysis of cell populations possible

Allows cell sorting on the basis of quantitative differences in the expression of cell surface markers

Statistically powerful Magnetic cell sorting

Fast, high-yield separation of cell populations

Suitable for large-scale sorting

Only for sorting, not for analysis

[7]

of cell populations

Sorting requires presence or absence of suitable cell surface markers

Multispectral imaging flow cytometry (MIFC)

Combines image acquisition and

Tissue cytometry

Automated and high-throughput

analysis with the statistical powers of flow cytometry

identification and analysis of cells in tissue sections or explanted tissues a)

Slower than flow cytometry No sorting option available yet No dynamic imaging

[8, 9]

Not applicable for intra vital imaging

[10]

or cell sorting

Identification, and in some cases, separation of cell subpopulations in suspension or in tissues, based on different criteria in each case

Each individual cell is measured as it flows past a laser beam giving information about its size, shape, granularity and the presence or absence of fluorescent labels within the cell or on the cell surface. The technology is not only extremely robust, allowing the analysis and sorting of thousands of cells per second, but is also very versatile; it has been applied to study cell cycle, cell viability, cell division and chromosome stability. Flow cytometry’s role in ‘‘bulk’’ cell sorting however, has been surpassed by magnetic cell sorting (Table 1), which uses antibodies conjugated to magnetic beads, thus allowing the separation of labeled and unlabeled cells in a magnetic field [7]. It not only allows the sorting of very large batches of cells, but is also much gentler than FACS, which exposes the cells to very high sheer forces during the separation process when run at high speed. Nevertheless, FACS remains one of the most powerful tools of immunologists. It excels when multiple markers are used to sort subpopulations of cells and is by far, the most versatile technology for the analysis of cell populations [6].

Ex vivo imaging The ability to isolate specific cell populations allowed the investigation of cell behaviors ex vivo, in defined and controlled


environmental conditions. Transmission light microscopy (Table 2) gives morphological information about transparent specimens [18] and can be used to observe the movement [19] and interactions of live cells [20]. In bright-field microscopy, the sample is simply illuminated from one side and observed from the other [11]. The contrast of the images is relatively low, but can be improved by using contrast enhancing techniques. Dark-field illumination collects only the light scattered by the sample, making images appear as bright objects on a dark background [11]. Phase contrast and Hoffman’s Modulation Contrast improve contrast by visualizing variations in the refractive index [13, 21]. The best contrast enhancement can be achieved using DIC, which converts variations in the optical density of the sample into variation in light intensity [12]. As the resolution of transmission light microscopy is limited by the wavelength of visible light, immunologists had to turn to other imaging approaches to obtain finer morphological information. Transmission electron microscopy (Table 2) works by the same principle as transmission light microscopy, but uses an electron beam and electromagnetic lenses to focus on a specific plane [22]. The spatial resolution achieved (which can be as high as 0.1 nm) is far greater than that of light microscopy (4200 nm), thus allowing imaging of the fine structure of cells at the level of organelles and macromolecules. This has contributed

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Table 2. Microscopy methods Advantages

Disadvantages

More information

Low contrast

[11]

Better contrast than bright-field Simple equipment set up

Requires strong illumination which

[11]

Higher contrast than dark-field

More costly than bright-field and dark-

Transmission light microscopya) Bright-field

Simplest and most affordable equipment set-up

Dark-field

Phase contrast

may damage biological samples

Differential interference contrast (DIC)

Highest contrast and resolution of all

Hoffman’s modulation contrast

Electron microscopyb) Transmission electron microscopy Scanning electron microscopy

Simpler and more affordable equipment

Requires more complex and expensive

High resolution (up to 0.1 nm)

Laborious sample preparation Not suitable for live cell studies

[14]

High resolution (1–20 nm) imaging of

Laborious sample preparation Not suitable for live cell studies

[14, 15]

Phototoxicity and photobleaching due

[16]

surface morphology Depth of field

Fast and simple acquisition

Phototoxicity and photobleaching

Multi-photon microscopy

b) c)

restricted to small area illuminated by laser Suitable for three-dimensional reconstruction of serial optical sections

Higher tissue penetration depth

a)

[11, 13]

achieved is not as good as with DIC

Confocal microscopy

[11, 12]

equipment set-up Not applicable for birefringent specimens (such as common plastic tissue culture dishes)

At high magnifications, the contrast

set up than DIC Can be applied on birefringent specimens

Fluorescence microscopyc) Wide-field epifluorescence

types of transmission light microscopy Depth of field Very few artifacts Can be used for relatively thick specimens

[11]

field Objects are often distorted by an artifactual surrounding halo

(hundreds of mm) Less phototoxicity and photobleaching than with confocal microscopy Especially suitable for live tissue imaging

to wide spread illumination In thick specimens image is blurred by light collected from out of focus regions

Relatively slow acquisition Limited penetration depth (tens of mm)

[16]

when used in tissue imaging

More expensive instrumentation Small field of vision thus not applicable

[17]

for non-invasive, whole-body imaging

Information on size, morphology, position, mortility. Resolution up to 200 nm. Applicable on transparent specimens (tissue sections and cell suspensions). Contrast varies depending on the type of microscopic imaging. Used for high resolution imaging of tissue or cell morphology. Used to locate fluorescently tagged molecules within cells or tissues. Suitable for live cell imaging.


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to our understanding of the fine detail of many immunological processes ranging from autophagosomes during antigen processing [23], cell–cell junctions during antigen recognition [24], intercellular nanotubes [25], to detailed insight into the membrane architecture of lymphocytes [26]. It has been applied to an equally great extent to image the micro-architecture of secondary lymphoid organs [27]. The scanning electron microscope (Table 2) produces images of a sample surface by scanning it with a high-energy electron beam [28]. Its resolution is lower than that of the transmission electron microscope, but provides greater depth of field and a good, three-dimensional representation of the surface morphology [15]. It has been used to compare the morphology of normal and neoplastic lymphocytes [29] and to follow the spreading of T cells upon activation [30]. The main disadvantage of electron microscopy is that the tissue sections or cell suspensions have to be fixated, dehydrated, embedded and stained, thus prohibiting live-cell imaging [31]. Fluorescence microscopy bridges the gap between transmission light microscopy and electron microscopy, as it allows the imaging of live cells on a molecular level (Table 2). Whilst initially developed for the detection of auto-fluorescence, nowadays it is used to detect a wide variety of fluorescent probes ranging from fluorophore-coupled antibodies [32] to fluorescent proteins [33]. The specimen is illuminated with light of a specific wavelength, which is absorbed by fluorophores, which then emit light of longer wavelengths and thus different colors. The use of fluorochromes and fluorescent proteins with distinct emission spectra allows simultaneous imaging of several molecules. Widefield epi-fluorescence excels through its simple and affordable setup and rapid acquisition of images, but is not without drawbacks. The specimen is flooded with excitation light, which potentially leads to the destruction of fluorophores (photobleaching) and may have phototoxic effects. As the light emitted from ‘‘in’’ and ‘‘out of focus’’ regions cannot be easily distinguished, the image is always slightly blurred. Whilst this imaging artifact can be counteracted to a degree by deconvolution processing, the exact localization of the signal remains very difficult [34]. These problems have been largely overcome by the introduction of confocal microscopy, which uses point illumination and a pinhole in front of the detector to eliminate out-of-focus emitted light [35, 36]. As only the light from the focal plane is collected, the resulting images are sharp and accurate, allowing the reconstruction of 3D images by stacking serial, optical xy sections in the z-plane. Furthermore, the problems of photobleaching and phototoxicity are restricted to a small area that is illuminated, allowing the imaging of multiple areas of the specimen. Whereas until recently, the resolution of fluorescence microscopy was restricted by the diffraction limit of light, novel nanoscopy techniques surpass this constraint. In near-field scanning optical microscopy (NSOM) (Table 3) the light is not focused through lenses but rather passes through a small aperture near the specimen, thereby limiting diffraction [46]. An alternative is to narrow the point spread function (PSF) by physical means. Stimulated emission depletion (STED) (Table 3) uses two laser beams, one to excite fluorophores at the center of the focal spot and the


HIGHLIGHTS

other to deplete fluorophores in the periphery [50]. Ground state depletion microscopy (GSD) (Table 3) [51] shifts the molecules located at the outer region of the focal spot into a metastable dark state. A more indirect approach is to detect the photons emitted from individual fluorescently labeled molecules and to calculate their exact position through the PSF. This approach requires low concentration of the fluorescently labeled molecules so that the individual PSF do not overlap. Photoactivated localization microscopy (PALM) (Table 3) uses light-induced photoactivation of only a few fluorescent molecules at a time [48] and stochastic optical reconstruction microscopy (STORM) (Table 3) uses photoswichable fluorophores [49]. The gradual improvement in our understanding of biological processes, using more and more advanced imaging techniques, can be illustrated by the example of T–cell–APC interactions during antigen recognition. The initial observation using transmission light microscopy was that during antigen recognition, motile cells arrest and form stable junctions [19, 52, 53]. Electron microscopy revealed the ultra-structure of such tight cell–cell junctions [53]. First mechanistic insight into this process came with the demonstration of TCR capping and reorientation of the microtubule organizing center toward the contact area using epi-fluorescence microscopy [54, 55]. However, the dynamic nature of immunological synapses only became apparent when the contact interface between the cells was reconstructed in 3D [56, 57] and when more specialized microscopic techniques were applied.

Specialized microscopic techniques The co-localization of the signals from two independent fluorophores is often used as an indicator of proximity. Due to the resolution limit of fluoresecence microscopy, this approach falls short of detecting true molecular interactions. However, this limitation can be overcome by utilizing Fo ¨rster resonance energy transfer (FRET) (often referred to as fluorescence resonance energy transfer) (Table 3). When spectrally overlapping fluorophores come into close proximity, energy is transferred from one fluorophore to the other, causing a change in emission spectrum [58]. This makes it possible to measure the interactions between fluorescently labeled molecules. FRET has been used to study TCR and peptide/MHC class II [59], as well as TCR–CD8 [60] and TCR–CD4 interactions [61] and BCR clustering during antigen recognition [62]. Bimolecular fluorescence complementation (BiFC) is a promising alternative approach for measuring molecular interactions (Table 3). It is based on detecting fluorescence emitted upon complementation between two nonfluorescent fragments of a fluorescent protein [41]. In contrast to FRET, which leads to a shift in the emission spectrum that is at times tricky to measure, BiFC only gives a signal when an interaction occurs. As there is a considerable lag phase between the occurrence of the interaction and the complementation of the two non-fluorescent units to a fluorescent protein, BiFC is not suited for dynamic measurements.

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Table 3. Specialized optical techniquesa) Techniques

Advantages

Disadvantages

Total internal reflection fluorescence (TIRF)

High resolution, live imaging of cell Complex equipment set-up

Fluorescence recovery after photobleaching (FRAP)

Quantitative measurement of

Fluorescence correlation spectroscopy (FCS)

Measurement of diffusion and of

More information [37]

surface events Single molecule imaging

Measurements are extremely

protein diffusion dynamics.

protein–protein interactions Higher sensitivity than FRAP

High statistical accuracy and

[38]

temperature sensitive

Sensitive to photobleaching Requires low fluorophore concen

[39]

trations

temporal resolution Fo¨rster resonance energy transfer (FRET)

Measurement and monitoring of

Bimolecular fluorescence complementation (BiFC)

Measurement and monitoring of

Chemical calcium indicators

FRET is often difficult to measure

molecular interactions in situ Suitable for dynamic measurements

[40]

and does not alway give a reliable indication for molecular interaction

Not suitable for dynamic measure- [41]

molecular interactions in situ Straightforward measurement compared to FRET

ments

Monitoring of intracellular calcium Chemical calcium indicators can levels

[42]

leak from the cells and are not ideal for long-term imaging

Simple usage No need for genetic manipulation of the cells Genetically encoded calcium indicators

Monitoring of intracellular calcium Need for genetic manipulation of

Optical tweezers

levels. Can be targeted to specific cellular compartments Stable expression allows long term imaging Transgenic animals allow in vivo imaging without ex vivo manipulation of cells

[42]

the cells Background from calmodulin binding to other cellular components Change in signal upon calcium binding is not very marked

Mechanical micro-manipulation of Complex equipment set-up microscopic objects Potential damage of biological Suitable for sorting cells in combi- material (opticution)

[43–45]

nation with microfluidics Near-field scanning optical microscopy (NSOM)

Resolution beyond light diffraction Not possible to image intracellular [46] limit

events

Technically demanding Slow acquisition Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)

Resolution beyond light diffraction Long data acquisition times (hours) [47–49] limit Requires photoactivatable probes Applicable with standard epifluor escence or confocal microscopy

Applicable for three-dimensional and live cell imaging


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Table 3. Cont. Stimulated emission depletion (STED)

Resolution beyond light diffraction Complex set-up. Requirement for limit No need for photoactivatable more specilized lasers.

[47, 50]

probes

Applicable for live cell imaging Ground state depletion microscopy (GSD)

Resolution beyond light diffraction Complex set-up. [47, 51] Requirement for more specialized limit No need for photoactivatable lasers. probes

a)

Optical techniques which are applied for specific assessments during live cell imaging

Many cellular signaling events lead to an increase in intracellular calcium levels. This can be monitored using chemical calcium indicators (Fig. 1A, Table 3), such as Fura-2 and Indo-1, which change their spectral emission properties upon binding of calcium ions [65]. This approach has been used to demonstrate that TCR activation-induced calcium flux leads to changes in the motility of T cells, allowing stable contact formation with APC [19] (Fig. 1A) and that TCR signaling is sustained during prolonged T-cell–APC contacts [66]. An alternative are genetically encoded calcium indicators, in which two spectrally shifted GFP mutants are linked by calmodulin and the calmodulinbinding peptide M13. In this case, an increase in the calcium concentration leads to FRET between the two fluorescent moieties [67]. The advantage of the latter method is that the indicators are genetically coded and therefore can be targeted to specific intracellular compartments [67]. Genetically encoded probes can also be applied to monitor other signal transduction events. In particular, FRET-based probes consisting of two GFP mutants linked by a kinase substrate and a phosphorylation recognition domain, have been used to measure protein phosphorylation [40, 68]. Similarly, probes consisting of two GFP mutants linked by a phosphoinositide-binding unit, have been used to monitor changes in lipid concentration [40, 69, 70]. Whilst photobleaching is generally an unavoidable nuisance accompanying fluorescence microscopy, in fluorescence recovery after photobleaching (FRAP) experiments (Fig. 1B, Table 3), photobleaching is exploited to measure the diffusion of specific molecules [38]. A defined area is bleached and the recovery of fluorescence resulting from the influx of labeled proteins from the non-bleached area is measured [71]. FRAP was used to demonstrate that the mobility of heterochromatin protein 1 within euchromatin and chromatin increases during T-cell activation [72]. Similarly, it was used to show that during synapse formation, persistent ZAP-70-containing TCR micro clusters are not static but continuously exchange ZAP-70 molecules [73]. Fluorescence correlation spectroscopy (FCS) (Table 3) provides a more sensitive approach to monitor protein diffusion [39, 74]. It measures fluctuations in fluorescence intensity that result from the diffusion of fluorescently labeled molecules in and out of a defined volume. Due to its high sensitivity, this method can detect protein–protein interactions as a


function of changes in the diffusion of free versus complexed molecules. FCS was used to reveal highly dynamic nanodomains of the plasma membrane of T cells that play a role in CD28 signaling [75]. Optical techniques can also be used to mechanically manipulate objects. Optical tweezers (Table 3) use a focused laser beam to exert small mechanical forces (in the pN range) on microscopic dielectric objects [76]. This technique, often referred to as optical trapping, has been used to manipulate contacts between natural killer cells and target cells [77] and to show polarity of antigen recognition by T cells during their initial contact with B cells [19]. It was used to orient cells during the imaging of immunological synapses, allowing the direct visualization of the contact interface between the cells [43], which yields much better results than 3D reconstruction from confocal image stacks. Optical tweezers can also be used to trap single molecules in order to image them for long periods of time [78]. The strength of ex vivo, microscopic imaging clearly lies in the qualitative assessment of biological processes, rather than large-scale quantitative analysis. Multispectral imaging flow cytometry (MIFC) captures images of cells in flow at a rate of hundreds of images per second (Fig. 1C, Table 1). The quality of the images is comparable to that obtained using wide-field epifluorescence microscopy. Although the technology is only a few years old, it is being rapidly adopted by immunologists. It has been used to quantitate nuclear translocation of the transcription factor NF-kB in mixed cell populations [8] and to show that NF-kB activation in T lymphocytes is regulated by the adaptor proteins ADAP and CARMA1 [9]. Further, MIFC has been used for quantitative analysis of protein co-localization in B cells [79] and for the separation of live, necrotic and apoptotic cells based on photometric and morphometric criteria [80].

Mimicking nature Planar substrates and supported lipid bilayers have been used to simplify the study of ligand–receptor interactions across cells and tissues (Table 4). In its most basic form, a planar surface is coated with a single type of protein [73, 85]. A more sophisticated imitation of interaction surfaces can be achieved with glasssupported lipid bilayers [81]. The composition of molecules within

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the bilayer can be easily manipulated and membrane proteins retain a high degree of lateral mobility. Lipid bilayers containing MHC class II and ICAM-1 have been used as surrogate ‘‘APC’’ membranes [86] to study the kinetics of synapse formation [87], TCR signaling [88] and the mechanisms of antigen acquisition by B cells [89]. They were also used to address the functional relevance of molecular segregation in T-cell activation [90]. Total internal reflection fluorescence (TIRF) microscopy (Fig. 1D, Table 3) has proven to be particularly useful for high-resolution visualization of the interactions of molecules in these cell-substrate contacts. It is based on the detection of evanescent waves, which are generated upon total reflection of the excitation light at the interface between a cover glass and the specimen. These waves only reach about 100 nm into the sample, avoiding background fluorescence excitation [37]. TIRF has been used to reveal that, during T-cell antigen recognition, TCR micro-clusters constitute the signaling components [88] and that specific cell surface receptors are either concentrated in or excluded from micro-domains within the T-cell membrane [64] (Fig. 1D). In combination with epi-fluorescence microscopy, it has been used to demonstrate that mitochondria within T cells translocate toward the vicinity of the immunological synapse [91]. Arguably, these very reductionist approaches simplify the biological context to its absolute minimum and are thus restricted in their application. Striving for a better mimic of the in vivo context, three-dimensional extracellular matrices have been developed to approximate the three-dimensional collagen networks of lymphatic tissues (Table 4). Despite promising initial results [92], progress in this direction has been surprisingly slow. Whilst ex vivo imaging techniques and reductionist approaches have proven to be extremely useful in gaining mechanistic insight into individual components of the immune system, doubt remains as to whether they reflect the true behavior of the cells in situ. Whilst the restriction of variables to the absolute minimum facilitates the dissection of processes, it is limited to known parameters and susceptible to artifacts. Thus, it is important to confirm the biological relevance of the findings obtained ex vivo using in vivo models or at least to complement them with in situ observations.

Tissue imaging Bright-field, high-contrast transmission light or epi-fluorescence microscopy have been used over several decades to gain insight in leukocyte trafficking in situ [93]. However, due to the lack of penetration depth, the observations were limited to transparent, thin, typically membranous tissues. Although epi-fluorescence allowed the identification of cells within thicker tissues, it was however hampered by phototoxicity and photobleaching [93]. The imaging of live cells in explanted immune tissues by confocal microscopy permitted realtime, three-dimensional observation of immunological processes in their natural micro-environment [94, 95]. However, this approach is not without technical limitations. The main issue is the limited tissue penetration of short wavelength (high energy) excitation lasers. Increasing their intensity is not feasible as this would lead to considerable tissue damage. Multi-photon microscopy (Fig. 2,


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Figure 1. Advanced optical techniques. (A) Intracellular calcium imaging. Image adapted from Fig. 4 of [19] with permission from Elsevier, copyright 1996. The original legend read: ‘‘Images of T–B cell contact and T cell [Ca21]i at various times following contact with B cells. Either naı¨ve or B cells presenting HEL peptide were settled onto a cover glass with adherent fura-2-loaded T cells.’’ (B) FRAP. Image adapted from Fig. 2 of [63] with permission from Macmillan Publishers Ltd, copyright 2000. The original legend read: ‘‘Qualitative FRAP analysis of ER-localized VSVGGFP in cells incubated at 40 or 321C in the presence of brefeldin A for 24 h. Images were obtained before photobleaching and at the indicated time points after. The photobleached area is outlined by a box.’’ (C) MIFC. Image kindly provided by Amnis Corporation. Example multispectral cell images from PMAstimulated murine peripheral lymph node cells. bright-field (white), NF-kB (green), 7-AAD (red), and NF-kB/7-AAD (green/red) composite images for each cell are shown. (D) TIRF microscopy. Image adapted from Fig. 6 of [64] with permission from Elsevier, copyright 2005. The original legend read: ‘‘Dual-Color Imaging of Single GFP-Tagged Molecules Relative to CD2-mRFP Clusters. A single frame from an image sequence of single molecules of Lck-GFP (green) that was superimposed upon a snapshot, bandpass-filtered image of CD2-mRFP clusters (red)’’.

Table 2) uses long wavelength (low energy) lasers that can penetrate tissues for hundreds of mm [97] and thus allows observation deeper in the tissue. In this case, excitation of the fluorophores is achieved by the simultaneous absorption of multiple photons, each contributing to the energy required for excitation. Like confocal imaging, multi-photon microscopy can be used to acquire z-stacks, allowing the assembly of 3D images. It was first used to visualize T cell–B cell interactions in explanted lymph nodes, confirming the ex vivo observation that in the presence of cognate antigen, the cells interact for longer periods of time [98]. It has since been applied in a wide range of studies including the imaging of CD4 [99] and CD8 T cell–dendritic cell interactions [100], germinal center formation [101, 102] and antigen delivery by macrophages [103]. A number of studies went one step further and performed true intra vital imaging in anesthetized mice by surgically exposing the tissue of interest. The first study using this approach revealed that, in contrast to

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Table 4. Special substrates, beyond microscopic slides, in which cells can be imaged Emulation of context

Advantages

Disadvantages

More information

Planar substrates and lipid bilayers

Simplification of ligand-receptor

Substrate does not always reflect physiological cell behavior

[81]

Substrate does not always reflect physiological cell behavior

[82]

Substrate does not always reflect physiological environment

[83, 84]

interactions across cells Especially suitable for high resolution imaging of cell-contact interface with TIRF

Three-dimensional extracellular matrices

Better imitation of the tissue envir-

Microfluidics

Miniaturization and integration of

onment than a glass surface Especially suitable for studies of cell migration and interaction

assays of choice in a chip format Suitable for high-throughput imaging of single cells or small organisms

monitoring of long range trafficking of cells and the study of low numbers of labeled cells is rendered all but impossible. Whilst the latter remains a problem to be solved, the former can be addressed or at least complemented by non-invasive, whole-body imaging.

Whole-body imaging Figure 2. Multi-photon microscopy. Image adapted from Fig. 7 of [96] with permission from Elsevier, copyright 2008. The original legend read: ‘‘CMTPX-labeled in vitro stimulated OT-II T cells (red) were transferred into LysM-EGFP mice (green) that had been infected with BCG-RFP bacteria 3 wk earlier. 12 h later, BSA-647 (white) was injected into the animals to visualize the sinusoidal network and hepatic IVM was performed. A single time point from a 4D data set showing T cells in relation to sinusoidal network (left), T cells in relation to macrophages (middle), and T-cell migration paths (blue) in relation to macrophages (right).’’

observations made with ex vivo cell populations, naı¨ve CD4 T cells are highly motile in situ and move in a random fashion even in the absence of TCR stimulation [104]. Whilst most intra vital imaging studies rely on the adoptive transfer of fluorescent-labeled cells, the use of transgenic mice expressing a fluorescent reporter in subsets of immune cells completely avoids ex vivo experimental manipulation [105]. More recently, intra vital, multi-photon imaging has been successfully combined with advanced fluorescent techniques, such as intracellular calcium imaging [106] and FRET [107]. The hope is that in future, multi-photon microscopy can be extended to tissue cytometry (Table 1) by combining automated image acquisition with automated tracking of individual cells [10], thus providing us with a robust technology for the acquisition of real-time, 3D data of immunological processes. A further complication of intra vital imaging is that in order to be imaged, the tissue has to be surgically exposed. This in itself might induce a local inflammatory response, thereby influencing any observations made. In addition, there are technical limitations. The field of vision is very small and as a result does not allow


Many of the currently used techniques for whole-body imaging were originally developed for diagnostic purposes in clinical settings. Positron emission tomography (PET) (Fig. 3A, Table 5) for example, can provide functional information of biological events through the use of molecular probes labeled with positron-emitting isotopes, such as 18F, 15O, 13N and 124I [115]. Interestingly, PET-scanners do not measure the positron emission, but detect the gamma radiation generated in the annihilation reaction between emitted positrons and nearby electrons. PET has been used to monitor the distribution of T cells in the body during anti-tumor responses [108, 116] (Fig. 3A) and to study the activation of T cells in vivo [117]. Whilst the approach excels through its very high sensitivity (detection limit of 10 11–10 12 M) and large variety of tracer probes, it has limited spatial resolution (1–2 mm). Magnetic resonance imaging (MRI) (Fig. 3B, Table 5) provides both specificity through the use of molecular probes and highresolution anatomical information (25–100 mm range) and is currently the only non-invasive approach, which can be used to monitor the immune system at a cellular level [118]. MRI uses a strong magnetic field to align the spin of hydrogen nuclei in water molecules within soft tissues. A pulse of radio waves shifts them into an excited state and the radio waves emitted during their relaxation to the baseline orientation are measured. Depending on their context, the hydrogen nuclei relax at different speeds, thereby providing image contrast and anatomical information. Specific labeling can be achieved with antibodies coupled to contrast agents such as paramagnetic cations, and super paramagnetic iron oxides [113]. MRI has been used to track low

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Figure 3. Whole-body imaging. (A) PET. Image adapted from Fig. 5 of [108] with permission from the National Academy of Sciences of the USA, copyright 2003. The original legend read: ‘‘(Right) An animal bearing the M-MSV/M-MuLV-induced tumor and P815 tumor was imaged by using [18F]FHBG. A stronger signal was detected in the antigen-positive tumor. The average of five planes that had the greatest signal in the region of interest is presented. (Left) For orientation, a picture of the tumor-bearing animal.’’(B) MRI. Image adapted from Fig. 3 of [109], with permission from the American Society for Clinical Investigation, copyright 2005. The original legend read: ‘‘Young female BDC2.5/NOD mice were imaged on day 6 after CPA treatment and 24 h after MNP injection. A pseudocolor was assigned to the pancreas, reflecting the T2 value of the organ. Photomicrographs show representative islet histology from these animals’’. (C) Bioluminescence imaging. Image adapted from Fig. 1 of [110] with permission from Macmillan Publishers Ltd, copyright 2003. The original legend read: ‘‘Distribution of T cells in BALB/c recipients 7 d after transplantation of luc1 Tconv cells with (right) or without (left) co-transplantation of CD41CD251 Treg cells. Color bar represents signal intensity code over body surface area.’’ (D) Imaging of zebrafish. Image adapted from Fig. 6 of [111] with permission from the National Academy of Sciences of the USA, copyright 2004. The original legend read: ‘‘Lymphopoiesis is fully reconstituted in irradiated adult recipients transplanted with lck-GFP kidney marrow. Transplants consisted of thymus cells (1.5 106 cells, A and B) or whole kidney marrow (3 105 cells, C and D) at 15 or 24 days posttransplantation. The views are lateral with anterior to the left. Arrowheads denote the location of the thymus (T).’’

Table 5. Whole-body imaging techniquesa) Technique

Advantages

Disadvantages

More information

Positron emission tomography (PET)

Large availability of molecular

Limited spatial resolution (1–2 mm) Expensive instrumentation

[112]

Low temporal resolution

[113]

Magnetic resonance imaging (MRI)

probes High sensitivity of detection High temporal resolution (seconds to minutes) Tomography allows 3D reconstruction

High spatial resolution (25–100 mm) Availability of molecular probes Tomography allows 3D

(minutes to hours) Expensive instrumentation

reconstruction Bioluminescence

High sensitivity of detection High temporal resolution (seconds to minutes)

Light transmission through

Simple and inexpensive set-up compared with PET and MRI

[114]

certain tissues types is limited Not yet applicable for human studies Two-dimensional information

Low background a)

Optical and non-optical imaging techniques with a large field of vision and penetration depth that allow non-invasive, whole-body imaging


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Eur. J. Immunol. 2009. 39: 1188–1202

numbers of magnetically labeled dendritic cells [119] and to monitor transplanted hematopoietic stem cells in immunodeficient mice [120]. However, the use of this technology is hampered by its high cost and the fact that the temporal resolution is inadequate for real-time dynamic imaging of rapid biological events. Much better temporal resolution can be achieved with Bioluminescence (Fig. 3C, Table 5), which is based on the detection of light emitted by enzymatic (typically renilla, firefly or bacterial luciferase) reactions. Charged coupled device cameras can be used for non-invasive, real-time tracking of cells in live animals. The approach is very sensitive with a low background. It can be used for whole-body imaging, whilst retaining the possibility of zooming into an area of interest to achieve cellular resolution [121]. It has already been used for a large number of different applications ranging from tracking the spread of an infection by tagging bacteria [122], to the monitoring of tumor cells during immunotherapy [123] and the tracking of immune cells in the context of transplantation (Fig. 3C) [110]. In vivo imaging at the tissue or whole-body level allows us to characterize how cells behave during an immune response. However, current intra vital and whole-body imaging techniques are difficult to scale up for genetic studies in mice, as they are expensive, labor intensive and technically demanding. Arguably, zebrafish (Fig. 3D) provide us with an animal model to circumvent such limitations. They possess components of both the innate and adaptive immune system, which share significant homology with those of higher vertebrates [124, 125]. The key advantage of zebrafish as a model system is its suitability for imaging. The free-living embryos are optically transparent, allowing high-resolution imaging of the immune system at developmental stages that are inaccessible in mammals [126]. Whilst adult zebrafish are not as transparent, their small size (approx. 3 cm) is advantageous for whole-body imaging. Immuno-histochemical staining has been used to follow neutrophils during successive developmental stages [127] and whole mount RNA in situ hybridization has been used to characterize the expression of hematopoietic candidate genes [128]. A transgenic zebrafish line expressing an lck-promoter-driven GFP has been used to follow the development of the T-cell lineage [111] (Fig. 3D) and photo-activatable cell tracers have been used to track the colonization of successive hematopoietic organs by hematopoietic precursor cells [129]. Most importantly, the fast and prolific reproduction of zebrafish makes them particularly suited for forward (random mutagenesis) [130] and reverse (targeted modification) genetic screens [131, 132].

HIGHLIGHTS

surrounding cells in studies that exceed an observation length of an hour. Furthermore, even if the populations are defined based on the presence of several lineage specific markers, it is unlikely that they will be truly homogeneous. This ‘‘black box’’ problem is even more prominent in intra vital and whole-body imaging. It should always be borne in mind that the observed labeled cells are not floating in black space, but rather squeeze past the much larger number of non-labeled and thus invisible cells of the surrounding lymphoid tissue. Indeed, the stromal micro-anatomy of the lymph nodes and spleen itself appears to play a central role in orchestrating cell migration [133, 134]. At the other extreme, the general consensus is that individual cells alone are not ‘‘happy’’; an insight gained from limiting dilution studies. This problem can be ameliorated by culturing them in ‘‘conditioned medium’’, i.e. the supernatant of cell populations [135, 136]. This suggests that the cells require endocrine factors to survive, which in the context of a single cell in a vast volume of medium becomes too dilute. The solution might be to drastically reduce the volume in which the cells are cultured, an approach that has been hampered by technical problems in handling and maintaining small volumes of liquid. The solution comes in the form of microfluidics (Fig. 4, Table 4). The underlying idea of this rapidly evolving technology is the miniaturization of standard laboratory techniques and their integration into ‘‘lab on a chip’’ devices using mirofluidic circuits. The technology has already been applied in genomics [138], proteomics [139], sequencing [140] and drug screening [141]. Whilst the implementation of microfluidics in immunological research is still mostly outstanding, working prototypes of the individual components already exist, making it possible to load, sort, culture, image and manipulate individual cells [83].

Microfluidics Whereas great advances have been made in the imaging of cell populations, it still remains challenging to image and study the interaction of and communication between individual cells. Ex vivo studies invariably deal with cell populations, making it all but impossible to completely separate the signals exchanged between the cells under observation from those emitted by the thousands of


Figure 4. Microfluidics. Image represents Fig. 1 of [137], reproduced with permission from the Royal Society of Chemistry, copyright 2006. The original legend read: ‘‘Single cell trapping arrays. (A) A photograph of the cell-trapping device is shown demonstrating the branching architecture and trapping chambers with arrays of traps. (B) A diagram of the device and mechanism of trapping is presented. (C) A high resolution brightfield micrograph of the trapping array with trapped cells is shown.’’

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1198


Microfluidic devices have already been used to monitor T-cell signaling [84] and to study T-cell migration [142]. The possibilities for the future are compelling. Microfluidic systems are likely to incorporate components such as magnetic cell sorting [143], FACS [144] and optical trapping [145]. In principle, any assay or experimental system can be miniaturized and customized for specific purposes. Indeed, a multitude of microbiological and chemical assay systems have already been miniaturized and can thus be readily coupled downstream of cellbased applications. Before long, we may be able observe and analyze the interaction between individual cells and monitor the consequences thereof on their transcriptional program using single molecule detection readouts.

Eur. J. Immunol. 2009. 39: 1188–1202

2 Gordon, S., Elie Metchnikoff: father of natural immunity. Eur. J. Immunol. 2008. 38: 3257–3264. 3 Bajenoff, M. and Germain, R., Seeing is believing: a focus on the contribution of microscopic imaging to our understanding of immune system function. Eur. J. Immunol. 2007. 37: S18–S33. 4 Hunt, V. and Weir, D., Cellular Immunology: Handbook of Experimental Immunology. Blackwell Scientific Publication Oxford 1978. 5 Kohler, G. and Milstein, C., Continuous cultures of fused cells secreting antibody of predefined specificity. 1975. J. Immunol. 2005. 174: 2453–2455. 6 Herzenberg, L., Parks, D., Sahaf, B., Perez, O., Roederer, M. and Herzenberg, L., The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin. Chem. 2002. 48: 1819–1827. 7 Thiel, A., Scheffold, A. and Radbruch, A., Immunomagnetic cell sorting – pushing the limits. Immunotechnology 1998. 4: 89–96.

Perspective Metchnikoff’s work stands out not only because of its scientific brilliance, but also because of his visionary approach to tackle complex biological problems. Based on his in situ observations, he proposed a general biological mechanism. He tested his hypothesis by ex vivo experimentation and then proceeded to confirm the biological relevance of his findings using in vivo models [1]. A 100 years later, we have a much larger toolbox of imaging techniques at our disposal. Each of them has its own advantages and limitations regarding resolution, background, sensitivity, efficiency, robustness, physiological relevance, scalability and suitability for experimental intervention. The challenge we face in our studies is not only to choose the most appropriate imaging technique for the question we are asking, but to integrate the findings from different ex vivo and in vivo model systems. It would be interesting to know what Metchnikoff would have done if he were to have had all of today’s technologies at his disposal. Perhaps he would have followed up his observations from genetic screens in zebrafish, with ex vivo experiments in a microfluidics system, confirmed them using intra vital microscopy in mice and completed his study with whole-body imaging in humans.

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Acknowledgements: The authors are funded by the Medical Research Council. The authors would like to thank Brad Amos, Felix Randow, Geoff Williams and Kristian Andersen for critical reading of the manuscript and helpful discussions. Conflict of interest: The authors declare no financial or commercial conflict of interest.

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Abbreviations: BiFC: bimolecular fluorescence complementation DIC: differential interference contrast FCS: fluorescence correlation spectroscopy FRAP: fluorescence recovery after photobleaching FRET: Fo¨rster resonance energy transfer MIFC: multispectral imaging flow cytometry MRI: magnetic resonance imaging PET: positron emission tomography PSF: point spread function TIRF: total internal reflection fluorescence Full correspondence: Dr. Alexander G. Betz, Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, UK Fax: 144-1223-412178 e-mail: [email protected]

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Received: 28/10/2008 Revised: 8/1/2009 Accepted: 12/12/2009

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