Intravital two-photon imaging of T-cell priming and tolerance in the lymph node.
ABSTRACT Two-photon microscopy makes it possible to image in real-time fluorescently labeled cells located in deep tissue environments. We describe a procedure to visualize the behavior of lymph node T cells during either priming or tolerance, in live, anesthetized mice. Intravital imaging of T lymphocytes is a powerful tool to study the cellular orchestration of adaptive immune responses in physiological settings. This method should provide new insights into the regulation of lymphocyte migration and cell-cell interactions in various immunological contexts.
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ABSTRACT: Interactions between T cells and dendritic cells (DCs) in the lymph nodes are crucial for initiating cell-mediated adaptive immune responses. With the help of two-photon imaging, the complexity of these cellular contacts in vivo has recently been captured in time-lapse movies in several immunological contexts. Well beyond the satisfaction of seeing a T-cell response as it happens, these experiments provide fundamental insights into the regulation and the biological meaning of T-cell-DC contact dynamics. This Review focuses on how this emerging field is changing our perception of T-cell activation by DCs.Nature reviews. Immunology 10/2008; 8(9):675-84. DOI:10.1038/nri2379 · 33.84 Impact Factor
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ABSTRACT: We constructed a multiphoton (2-P) microscope with space to mount and operate microphysiology hardware, and still acquire high quality 2-P images of tumor cells deep within tissues of live mice. We reconfigured for nondescanned 2-P imaging, a dedicated electrophysiology microscope, the Nikon FN1. This microscope is compact, with retractable objectives, allowing more stage space. The instrument is fitted with long-working-distance objectives (2.5- to 3.5-mm WD) with a narrow bore, high NA, and efficient UV and IR light transmission. The system is driven by a powerful 3.5-W peak power pulsed Ti-sapphire laser with a broad tuning range. This 2-P system images a fluorescent standard to a depth of 750 to 800 microm, acquires images of murine pancreatic tumors in vivo, and also images fluorescently labeled T-cells inside live, externalized mouse lymph nodes. Effective imaging depths range between 100 and 500 microm. This compares favorably with the 100- to 300 microm micron depth attained by many 2-P systems, especially descanned 2-P instruments, and 40-microm-deep imaging with confocal microscopes. The greater depth penetration is attributable to the use of high-NA long-working-distance water-dipping lenses incorporated into a nondescanned instrument with carefully configured laser beam introduction and image-acquisition optics. Thus the new system not only has improved imaging capabilities, but allows micromanipulation and maintenance of tissues and organs.Journal of Biomedical Optics 01/2009; 14(2):024032. DOI:10.1117/1.3103783 · 2.75 Impact Factor
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ABSTRACT: In vitro studies have been essential to describe the molecular details of bacteria-host cell interactions in general and the functions of bacterial effector proteins in particular. Recent advancements in in vivo imaging techniques are facilitating the next logical step to visualize the dynamic infection process as it happens within the living host while analyzing the role of bacterial effector proteins in vivo. Data obtained from this emerging field of 'tissue microbiology', combined with the massive knowledge base generated from research in 'cellular microbiology' will eventually provide a complete picture of the complex infection process.Current opinion in microbiology 02/2009; 12(1):31-6. DOI:10.1016/j.mib.2008.11.002 · 7.22 Impact Factor