{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T14:47:21Z","timestamp":1791298041067,"version":"4.1.0"},"reference-count":18,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5574","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2002,6,7]]},"abstract":"<jats:p>Lymphocyte motility is vital for trafficking within lymphoid organs and for initiating contact with antigen-presenting cells. Visualization of these processes has previously been limited to in vitro systems. We describe the use of two-photon laser microscopy to image the dynamic behavior of individual living lymphocytes deep within intact lymph nodes. In their native environment, T cells achieved peak velocities of more than 25 micrometers per minute, displaying a motility coefficient that is five to six times that of B cells. Antigenic challenge changed T cell trajectories from random walks to \u201cswarms\u201d and stable clusters. Real-time two-photon imaging reveals lymphocyte behaviors that are fundamental to the initiation of the immune response.<\/jats:p>","DOI":"10.1126\/science.1070051","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T05:54:32Z","timestamp":1027749272000},"page":"1869-1873","source":"Crossref","is-referenced-by-count":1061,"title":["Two-Photon Imaging of Lymphocyte Motility and Antigen Response in Intact Lymph Node"],"prefix":"10.1126","volume":"296","author":[{"given":"Mark J.","family":"Miller","sequence":"first","affiliation":[{"name":"Department of Physiology and Biophysics,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sindy H.","family":"Wei","sequence":"additional","affiliation":[{"name":"Department of Physiology and Biophysics,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ian","family":"Parker","sequence":"additional","affiliation":[{"name":"Department of Neurobiology and Behavior, University of California, Irvine, CA 92697\u20134561, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael D.","family":"Cahalan","sequence":"additional","affiliation":[{"name":"Department of Physiology and Biophysics,"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1006\/smim.1999.0163"},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.immunol.19.1.375"},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0952-7915(00)00217-X"},{"key":"e_1_3_1_5_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.immunol.19.1.23"},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1054\/ceca.2001.0246"},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0896-6273(00)81237-4"},{"key":"e_1_3_1_8_2","unstructured":"BALB\/c CD3 + T lymphocytes from peripheral lymph nodes and na\u0131\u0308ve BALB\/c CD19 + B lymphocytes from the spleen were enriched by negative selection to >98% purity by magnetic depletion using anti\u2013MHC II MicroBeads (MHC major histocompatibility complex) or anti-CD43 MicroBeads respectively (Miltenyi Biotec Auburn CA). For purification of DO11.10 CD3 + T cells a combination of anti\u2013MHC II MicroBeads and DX5 anti\u2013natural killer cell MicroBeads was used as above. Cells were stained for 30 min at 37\u00b0C with 2 \u03bcM CFSE or CMTMR destained and washed with serum-free medium. Cells were resuspended in phosphate-buffered saline and adoptively transferred by tail vein injection into recipient BALB\/c mice 4 to 6 weeks of age. For cell-tracking experiments 10 million to 12 million purified CFSE-labeled T or B lymphocytes were adoptively transferred. Lymph nodes were harvested from the recipient mice 18 to 20 hours after adoptive transfer and were mounted in the imaging chamber. To observe the reticular fiber network we counterstained lymph nodes in 2 \u03bcM CMTMR in serum-free medium for 45 min at room temperature and then destained and washed them. The labeling was stable for at least 1 hour under our imaging conditions."},{"key":"e_1_3_1_9_2","unstructured":"Two-photon imaging was done with a custom-built video-rate multiphoton microscope (5) using an upright Olympus BX50 microscope fitted with a \u00d720 water-immersion objective (numerical aperture = 0.95) a Spectra-Physics Tsunami femtosecond laser and a resonant-mirror scan head. Image acquisition a z -axis stepper shutters and an emission filter wheel were under software control (MetaMorph Universal Imaging Downingtown PA). Lymph nodes were cemented to the base of the imaging chamber with a thin film of veterinary-grade superglue and were continuously superperfused with warm (35\u00b0 to 37\u00b0C) RPMI 1640 medium bubbled with 95% O 2 and 5% CO 2 . For imaging lymphocyte motility 5D image stacks ( x y z time and emission wavelength) were acquired as follows. Each xy plane spanned 200 \u03bcm by 200 \u03bcm at a resolution of 2 pixels \u03bcm \u22121 and was formed by averaging nine video frames. Alternate images at each plane were acquired at emission wavelengths of 510 to 550 nm (for CSFE-labeled cells) and 600 to 700 nm (for CMTMR-labeled cells) selected by a computer-controlled filter wheel. For each time point (10-s intervals) a stack of 17 two-color planes was acquired at axial ( z ) spacings of 3 \u03bcm with a computer-controlled focus motor. After recording image stacks were processed to yield maximum-intensity projections representing \u201ctop\u201d ( xy ) or \u201cside\u201d ( xz ) views through the scanned volume of the lymph node. For single-label experiments with CFSE excitation at 780 nm provided an optimal signal. For dual-label experiments the laser was tuned to 820 nm so as to equally excite both fluorophors."},{"key":"e_1_3_1_10_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.2125367"},{"key":"e_1_3_1_11_2","unstructured":"For antigenic challenge recipient BALB\/c mice were primed by subcutaneous injections with 150 \u03bcl of a 1.3% alum emulsion containing 3 \u03bcg of ovalbumin and 600 to 800 ng of murine tumor necrosis factor\u2013\u03b1 (Stemcell Technologies Vancouver BC). About 50 \u03bcl were injected into three sites on each mouse that were chosen to enhance the delivery of antigen to draining cervical and inguinal lymph nodes. DO11.10 T cells were purified and labeled with CFSE and 10 million to 12 million CD3 + T cells were adoptively transferred either into antigen-primed mice 24 hours after antigenic challenge or into unprimed recipient mice. At 1 and 5 days after adoptive transfer the nodes of antigen-primed animals and of unprimed control animals were placed side by side in the recording chamber for sequential imaging."},{"key":"e_1_3_1_12_2","doi-asserted-by":"crossref","unstructured":"M. Gunzer et al. Immunity 13 323 (2000).","DOI":"10.1016\/S1074-7613(00)00032-7"},{"key":"e_1_3_1_13_2","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-0029(19981201)43:5<369::AID-JEMT3>3.0.CO;2-6"},{"key":"e_1_3_1_14_2","first-page":"617","volume":"82","author":"Friedl P.","year":"1994","unstructured":"Friedl P., Noble P. B., Shields E. D., Zanker K. S., Immunology 82, 617 (1994).","journal-title":"Immunology"},{"key":"e_1_3_1_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1074-7613(00)80409-4"},{"key":"e_1_3_1_16_2","doi-asserted-by":"publisher","DOI":"10.1093\/intimm\/7.4.567"},{"key":"e_1_3_1_17_2","doi-asserted-by":"publisher","DOI":"10.1084\/jem.192.10.1425"},{"key":"e_1_3_1_18_2","doi-asserted-by":"publisher","DOI":"10.1085\/jgp.89.3.405"},{"key":"e_1_3_1_19_2","unstructured":"We thank A. Hejazi for help with cell tracking L. Forest for care and breeding of the DO11.10 mouse strain and G. Gutman and G. Chandy for helpful suggestions and comments on the manuscript. Supported by NIH grants GM-48071 (I.P.) and GM-41514 (M.D.C.). Supporting Online Material www.sciencemag.org\/cgi\/content\/full\/1070051\/DC1 Movies S1 to S6"}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.1070051","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,9]],"date-time":"2024-01-09T22:50:32Z","timestamp":1704840632000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.1070051"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2002,6,7]]},"references-count":18,"journal-issue":{"issue":"5574","published-print":{"date-parts":[[2002,6,7]]}},"alternative-id":["10.1126\/science.1070051"],"URL":"https:\/\/doi.org\/10.1126\/science.1070051","relation":{"has-review":[{"id-type":"doi","id":"10.3410\/f.1006984.92757","asserted-by":"object"},{"id-type":"doi","id":"10.3410\/f.1006984.86706","asserted-by":"object"},{"id-type":"doi","id":"10.3410\/f.1006984.90566","asserted-by":"object"}]},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2002,6,7]]}}}