In vivo Multiphoton Microscopy Technique to Reveal the Physiology of the Mouse Uterus 1 Ana C. Zenclussen 1 , David N. Olivieri 2 , Michael L. Dustin 3 , Carlos E. Tadokoro 4 1 Department of Experimental Obstetrics and Gynecology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany; 2 Escuela Superior de Ingenier  ıa Informatica, University of Vigo, Vigo, Spain; 3 Skirball Institute of Biomolecular Medicine, NYU Langone Medical Center, New York, NY, USA; 4 Laboratory of Immune Regulation, Instituto Gulbenkian de Ci^ encia, Oeiras, Portugal Keywords Dendritic cell, immunology, intravital imaging, multiphoton microscopy, uterus Correspondence Carlos E. Tadokoro, Instituto Gulbenkian de Ci^ encia, Rua da Quinta Grande, 6, Oeiras 2780-156, Portugal. E-mail: ctadokoro@igc.gulbenkian.pt Submission October 29, 2012; accepted November 28, 2012. Citation Zenclussen AC, Olivieri DN, Dustin ML, Tadokoro CE. In vivo multiphoton microscopy technique to reveal the physiology of the mouse uterus Am J Reprod Immunol 2012. doi:10.1111/aji.12066 Problem Pregnancy is a challenge to the maternal immune system as it allows the growing of a semiallogeneic fetus within the uterus. Such tolerance suggests a set of complex cellular distributions and interactions inside the organ. Until now, direct observation of such processes was absent because proper intravital imaging techniques were not available. Method We developed a new two-photon microscope stage together with a set of surgical procedures to provide direct observation of immune cell within the mouse uterus. Results Using our technique, we observed an accumulation of dendritic cells (DCs) in the uterus during the estrus phase of the estrus cycle. Some of the observed DC clusters were located near the lumen of the uterus or small blood vessels, each situated on the antimesometrium side. Conclusion While two-photon microscopy has become a widely used technology for intravital imaging, new advances in the development of staging and experimental protocols can still push the limits of this technique for exploring new biology. As proof of this, we demonstrated that with spe- cially designed staging and surgical protocols, we observed the formation of DC clusters in the uterus; structures that may play a role in the com- plex immunology of the uterus–fetal interface. Introduction For offspring to remain viable, cells of the immune system should maintain the basic functionality of the host tissue. These immune cells must exert spe- cialized functions while operating under tight regu- lation. 1 A failure of this cellular regulation can compromise the function of organs, potentially lead- ing to the collapse of the entire organism. 2 In preg- nancy, for example, a strong unregulated immune response against either the offspring or the repro- ductive system could compromise the mother’s future reproduction capability. 3 The maternal immune system experiences unique changes during pregnancy to simultaneously main- tain an effective defense against pathogens, poten- tially dangerous to the mother and develop immune tolerance against the semiallogeneic fetus that is growing in the uterus. Although there is direct con- tact between blood-maternal immune cells and fetal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 American Journal of Reproductive Immunology (2012) ª 2012 John Wiley & Sons A/S 1 TECHNICAL REPORT A J I 1 2 0 6 6 B Dispatch: 10.12.12 Journal: AJI CE: Nivetha Raj Journal Name Manuscript No. Author Received: No. of pages: 9 PE: Sangeetha