Two-photon fluorescence microscopy is a powerful research tool that employs ultra-fast pulsed lasers in combination with advanced optical laser scanning techniques to capture high-resolution three-dimensional images of fluorescently tagged specimens within intact tissues samples over time. This research methodology has the advantage of direct visualization of cellular arrangements and events that are often hard to deduce through sole use of in vitro assays and frozen sections, and the added benefit of being able to quantify many aspects of these events. As such, it is particularly well suited to address questions pertaining to the study of dynamic processes in living cells and tissues including, but not limited to, cell interactions, cell differentiation, and cell migration. Such studies can be conducted within virtually any tissue type; given tissue preparations can be developed to overcome the obstacles associated with maintaining native tissue structure and perfusion.
The In Vivo Microscopy Core Research Facility provides support for Yale investigators interested in utilizing two-photon fluorescence microscopy to achieve their research goals. The Core is responsible for novel protocol and methods development and is designed to serve as a clearinghouse for expertise and experience, being able to translate newly successful approaches to multiple projects and investigators. It is the Core’s goal to assist investigators in experimental design and implementation and to provide training for those whom wish to learn surgical preparations, imaging acquisition, and data analysis at the facilities image workstation utilizing Improvision’s Volocity software.
As an additional part of the educational outreach of this Core, we have, in collaboration with the Department of Cell Biology, put on annual hands-on Microscopy Workshops.
The imaging facility, which includes a dark imaging room and an antechamber for specimen preparations, is located in The Anlyan Center, in room S-614. The imaging workstation is located just down the hall outside of S-600.
The In Vivo Imaging Facility’s microscope suite is comprised of an antechamber equipped with a sink, lab bench, and dissecting microscope for specimen preparation, and a dark imaging room that houses the microscope. Wall mounted inhalation anesthesia (isoflurane) is available in the dark imaging room to enable longer, safer imaging.
LaVision Biotec TriMScope
The facility upright, laser scanning, two-photon microscope is operated with a tunable (680nm – 1080nm) Titanium-Sapphire Laser (Chameleon Vision II, Coherent) purchased in January 2010. The microscope is outfitted with a water immersion, long-working distance Olympus 20X objective lens (NA 0.95) and is capable of imaging both fixed and live specimens. Currently, fluorescent emission from four colors (Far-red, Red, Green, Blue) can be detected simultaneously from a sample utilizing non-descanned photomultiplier tubes (PMTs).
Macintosh Computer Workstation
The Mac Pro facility computer workstation is located just down the hall from the microscope suite and is available for data analysis utilizing Imrovision’s Volocity software. Volocity utilizes a variety of tools that allow for measurements in both three and four dimensions and the generation of high-resolution images/movies.
All of the facilities services are offered on a fee-for-service basis (see table below) with the exception of advise concerning experimental design, which is offered free of charge and which the Core strongly encourages users to utilize. Additional services will be added based on user demand and the further development of facility expertise and experience.
Training sessions for individuals are available for any of the approved tissue preparations, use of the two-photon microscope, and for data analysis utilizing Volocity (Improvision) or Imaris (Bitplane) software on the facility imaging workstation. Before individuals are allowed to operate any of the facility equipment or perform surgical preparations and analysis independently they must be observed and signed off by the facility manager to ensure that the equipment is not being abused or damaged and that the animals are being handled safely and humanely. This extra measure of precaution is because the facility operates with a single microscope, a single laser, and a single workstation and should any one of the pieces of equipment become compromised than ALL of the facilities users will suffer.
Current user fees are available here.
All new facility users need to provide the following information in order to receive a username and password for ScheduleBook, the online scheduling system for reserving the facility. ScheduleBook allows you to reserve time in the facility preparation room, on the two-photon microscope, and at the facility image analysis workstation.
- your name
- your contact information
- the PI's full name
- valid PTAEO
- proof of completion of the Laser Safety Online Training Course provided by OEHS
Please note: If you are reserving time during which you will require assistance from the facility technician, David Gonzalez, please be sure to communicate with him via e-mail prior to scheduling time in the facility.
STARTING JANUARY 2013
An individual lab may only reserve 8 days a month in advance during peak facility hours (8am-5pm, Monday - Friday) and should restrict their scheduling, as much as possible, to 2 days of peak time per week.
We understand that there may be an occasion where more than 2 days per week are needed. We are currently developing a policy to address such a circumstance.
If on Friday morning by 9:00am of a given week there is still peak-time available the following week, then anyone is free to book this time and it will not count towards the previously noted 8 day advanced booking limit.
Users are strongly encouraged to book only the time during which they will be imaging. For example, do not book the entire day (8am-5pm) if you do not plan on starting imaging until noon. Please book appropriately to free up additional imaging time for another user.
There will be no changes to the current user fees. All users are urged to take advantage of the 50% discount for all imaging conducted during off-peak hours and on the weekends.
Grants obtained using preliminary data generated in Core facility
- Bockenstedt (5R01AI085798-02 “Real-time imaging analysis of vector-borne Lyme Borreliosis pathogenesis and persistence”)
- W. Shlomchik (2R01HL083072-06 “Role of tissue antigen presenting cells in GVHD”)
- Walther Mothes R01 (5R01AI084096-03 “Targeting HIV cell-to-cell transmission”)
- Joe Craft (5R01AR040072-22 “Immune responses in Lupus”)
- Haberman (5R01AI080850-03 “In vivo imaging of T and B cell interactions in germinal center initiation”)
Publications resulting from Core C experimentation
- Park, J., S.H. Wrzesinski, E. Stern, J. Criscione, M. Look, S.M. Jay, S.L. Demento, A. Agawu, P.L. Limon, A.F. Ferrandino, D.G. Gonzalez, A.M. Haberman, R.A. Flavell, and T.M. Fahmy. 2012. Nanolipogel combination delivery of small molecule inhibitors and cytokine enhances tumor immunotherapy. Nature Materials (accepted).
- Bockenstedt, L.K., D.G. Gonzalez, A.M. Haberman, and A.A. Belperron. 2012. Intravital microscopy reveals inflammatory spirochete antigens persisting by cartilage after antibiotics for murine Lyme borreliosis. J Clin Immunol (accepted).
- Eisenbarth, S.C., A. Williams, O.R. Colegio, H. Meng, T. Strowig, A. Rongvaux, J. Henao-Mejia, C.A. Thaiss, S. Joly, D.G. Gonzalez, L. Xu, L.A. Zenewicz, A.M. Haberman, E. Elinav, S.H. Kleinstein, F.S. Sutterwala, and R.A. Flavell. 2012. Nlrp10 is a novel NOD-like receptor essential in initiating adaptive immune responses by dendritic cells. Nature 484: 510-515.
- Rompolas, P., D.G. Gonzalez, A.M. Haberman, and V. Greco. 2012. Two-Photon Microscopy to Capture Live Cell Behavior in the Hair Follicle Stem Cell Niche. Nature (accepted).
- Truman, L.A., K.L. Bentley, E.C. Smith, S.A. Massaro, D.G. Gonzalez, A.M. Haberman, M. Hill, D. Jones, W. Min, D.S. Krause, and N.H. Ruddle. 2012. ProxTom Lymphatic Vessel Reporter Mice Reveal Prox1 Expression in the Adrenal Medulla, Megakaryocytes, and Platelets. Am J Pathol 180: 1715-1725.
- Kerfoot, S.M., G. Yaari, J.R. Patel, K.L. Johnson, D.G. Gonzalez, S.H. Kleinstein, and A.M. Haberman. 2011. Germinal center B cell and T follicular helper cell development initiates in the interfollicular zone. Immunity 34:947-960.
- Esplugues, E., S. Huber, N. Gagliani, A.E. Hauser, T. Town, Y.Y. Wan, W. O'Connor, Jr., A. Rongvaux, N. Van Rooijen, A.M. Haberman, Y. Iwakura, V.K. Kuchroo, J.K. Kolls, J.A. Bluestone, K.C. Herold, and R.A. Flavell. 2011. Control of TH17 cells occurs in the small intestine. Nature 475:514-518.
- Goodwin, J.E., J. Zhang, D. Gonzalez, S. Albinsson, and D.S. Geller. 2011. Knockout of the vascular endothelial glucocorticoid receptor abrogates dexamethasone-induced hypertension. J Hypertens 29:1347-1356.
- Lee, H.K., M. Zamora, M.M. Linehan, N. Iijima, D. Gonzalez, A. Haberman, and A. Iwasaki. 2009. Differential roles of migratory and resident DCs in T cell priming after mucosal or skin HSV-1 infection. J Exp Med 206:359-370.
- Hauser, A.E., T. Junt, T.R. Mempel, M.W. Sneddon, S.H. Kleinstein, S.E. Henrickson, U.H. von Andrian, M.J. Shlomchik, and A.M. Haberman. 2007. Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns. Immunity 26:655-667.
Please note that the log sheet in the facility has been revised to reflect IACUC standards. There are now 4 separate boxes that require the user's initials, indicating the user has:
- wiped down the procedure room counter top and restraint platform with 70% ethanol;
- placed all sharps in the sharps container;
- placed all trash in the trash can;
- vacuumed up hair and bedding from the counter top and floor.
Users must continue to log time in and time out of the facility during a scheduled imaging study. This record keeping is MANDATORY per the IACUC.
Users will find the log sheet in a black binder at the imaging computer.
If there are any questions, please don't hesitate to ask Dave Gonzalez.
Details on a discount incentive for users willing to book "off-peak" imaging time in the facility are available here.