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

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. Twophoton 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.Scientific questions drive the development of technology, and new technologies, in turn, influence the type of questions that we pose in an iterative process of discovery. Applied to immunology, this has prompted the innovation and application of powerful techniques for analysing the microscopic world of the immune system. Two methodological lines of investigation have dominated the field: in vivo experiments that examine the behaviour of populations of cells in living animals during an immune response; and in vitro experiments that use individual cells in artificial environments. An immune response is the sum of many complex and dynamic individual cellular behaviours that are shaped by many environmental factors. In vivo experiments maintain this natural environment, but they cannot resolve the behaviours of individual cells. By contrast, in vitro experiments provide information at the subcellular and molecular levels, but they cannot replicate adequately the full repertoire of environmental factors. There is a pressing need for techniques that allow real-time observation of single cells and molecules in intact tissues. Recent developments in imaging technology now make this possible. In this review, we provide a guide to the application of biophotonic techniques to the field of immunology and, in particular, to the use of two-photon laser microscopy. We discuss recent results obtained using this approach and pinpoint some key questions that might be resolved by tissue imaging. The biophotonics tool kitImmunologists have long been adept at raiding other disciplines to acquire new research tools. Fluorescence techniques -for example, flow cytometry, video imaging and more exotic imaging modalities, such as FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) and two-photon microscopy -combined with new probes to track Ca 2+ signalling, Correspondence to M.D.C. mcahalan@uci.edu. NIH Public Access Imaging T-cell dynamics in vitroThe single-cell approach has intrinsic value, because asynchronous behaviours are not indicated by population measurements. The need for live-tissue imagingIn vivo approaches have elucidated the basic properties of immune cells and lymphoid tissues, whereas in vitro cell-culture systems have provided a high-definition analysis of the cellsurface receptors and intracellular signalling pat...

Characterizing T Cell Movement within Lymph Nodes in the Absence of Antigen

“…Two-photon microscopy has allowed the direct visualization of the movement of T cells within lymph nodes, giving new insights into immune interactions (1)(2)(3)(4). From these experiments, the locations of individual T cells moving in lymphoid organs are obtained as a function of time (5)(6)(7)(8).…”

“…Explanted lymph nodes are maintained at 36°C under superfused medium bubbled with 95% O 2 and 5% CO 2 to take account of the fact that the lymphoid environment is thought to operate at a relatively low oxygen tension (2). But despite these precautions, it remains a The parameter t pause was varied from 0 to 3.5 min, t free from 0.5 to 20 min, and v free from 5 to 50 m/min.…”

“…that explanted lymph nodes are deprived of their normal blood and lymphatic circulations, and this might have an effect on the experimental results obtained (2,3). Additionally, the four experiments were conducted using a variety of lymph nodes (e.g., popliteal, cervical, inguinal).…”

Two‐photon analysis of calcium signals in T lymphocytes of intact lamina propria from human intestine

“…Activation of NF-jB induces the expression of many pro-inflammatory cytokines that act locally within the intestinal microenvironment and cause inflammation [13,14]. To preserve the microenvironment and potential cell-cell interactions, we applied two-photon technology [15,16] [10]. In addition, analyzing a large number of cells from seven different donors, we found the same difference in the [Ca 2+ ] i plateaus between CD4 + and CD8 + PB T cells as described before [10].…”

3D and 4D imaging of immune cells in vitro and in vivo

“…Using high-intensity infrared laser-radiation, which can travel undiVractedly deeply into tissue, as a source for the excitation of Xuorophores, this technique allows visualizing Xuorescently labeled cells at high resolution, both in space and time. A very good review on the technical background of 2PM has been written by Cahalan et al (2002).…”