The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation Craig N. Jenne1,2, Connie H. Y. Wong1,3, Bjo¨ rn Petri1,3, Paul Kubes1,3* 1 Calvin, Phoebe & Joan Snyder Institute for Infection, Immunity & Inflammation, University of Calgary, Calgary, Alberta, Canada, 2 Department of Critical Care Medicine, University of Calgary, Calgary, Alberta, Canada, 3 Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada Abstract Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation. Citation: Jenne CN, Wong CHY, Petri B, Kubes P (2011) The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation. PLoS ONE 6(9): e25109. doi:10.1371/journal.pone.0025109 Editor: Dominik Hartl, Ludwig-Maximilians-Universita¨t M u¨nchen, Germany Received June 27, 2011; Accepted August 24, 2011; Published September 19, 2011 Copyright: 2011 Jenne et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Canadian Institutes of Health Research (http://www.cihr-irsc.gc.ca/) (C.H.Y.W. and P.K.), the Canada Research Chairs Program (http://www.chairs-chaires.gc.ca/) (P.K.), the Alberta Heritage Foundation for Medical Research (http://www.ahfmr.ab.ca/) (C.N.J and P.K.) and the Canadian Association of Gastroenterology (http://www.cag-acg.org/) (C.H.Y.W), and the Snyder Mouse Phenomics Core (http://www.iii.ucalgary.ca/) (B.P.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pkubes@ucalgary.ca Introduction The interface between innate and adaptive immunity is critically central to the host immune response. In recent years, platelets have emerged as an important component of this interface, shaping both the innate and adaptive immune responses [1–4]. Although primarily associated with hemostasis, platelets express a wide variety of immune receptors (TLRs, CD14, CD40, FcR) and release numerous immune effectors upon activation (IL1b, TGFb, RANTES, MCP-1, CD40L) [1]. Together, these receptors and molecules allow platelets to modulate leukocyte recruitment [5], activation and effector functions such as phagocytosis, cytotoxicity and extracellular DNA release (NETs) [6], key events at the crossroads between innate and adaptive immunity. A summary from the Fourth Annual Platelet Collo- quium concluded that platelets can recruit cells to sites of vascular injury, release pro- and anti-inflammatory factors, produce microparticles, and can induce thrombin generation [7]. These processes have been shown to be critical in the induction or progression of diseases such as atherosclerosis, sepsis, hepatitis, vascular restenosis, acute lung injury and transplant rejection. Although some information about platelet localization can be gained from immunohistochemical staining of tissue sections or radioisotope labelling of adoptively transferred platelets [2,8], the limitation of these techniques is the inability to study the dynamic interactions between platelets, leukocytes, and the vasculature. The development of intravital microscopy, and more recently, intravital spinning-disk confocal microscopy, has allowed for the observation of cellular behaviour within the tissue microenviron- ment of live animals. The capacity of spinning-disk confocal microscopy to capture individual frames of multiple fluorescence wavelengths in rapid succession, something that is not possible with conventional scanning-confocal microscopy, affords the ability to image rapidly moving particles, such as platelets, and makes this technology ideal for the study of platelet dynamics in vivo. The difficulty in studying platelets however, has been the identification of markers that can be used in vivo to specifically label platelets without affecting their behaviour. Currently, most studies have utilized one of two general approaches; either intravenous administration of a non-specific fluorescent dye such as rhodamine 6-G [9,10] or the adoptive transfer of platelets isolated from a donor animal, labelled in vitro with a fluorescent dye, and injected into a recipient animal [8,11]. These techniques allow for the visualization of platelets within the live animal, and have provided insight into platelet recruitment and adherence; however, they too have limitations. Although rhoda- mine 6-G efficiently labels platelets in vivo and allows for visualization of platelet-vessel wall interactions, due to its non- PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25109