Visualizing Dendritic Cell Networks In vivo Lindquist RA et al., Nat Imm. 5,12 (2004). Mike Leibowitz Exp Basis of Immunology January 10th, 2007

Previous work 2 photon microscopy was used to investigate cellular contacts involved in T cell development1 and the dynamics of CD8+ T cell activation by mature DCs in the lymph node2 The dynamics of DC mediated CD4+ T cell activation in the lymph node using 2 photon microscopy3 1Bousso P et al. Science. 296 (5574) 2002. 2Bousso P et al. Nat Imm. 4 (6) 2003. 3Miller MJ et al. J Ex Med. 200 2004.

Hypothesis ??? Aim- Interested in studying the activity of steady-state and mature dendritic cells (DCs) in lymph nodes of live mice using 2 photon microscopy

Dendritic Cells APCs DCs in periphery Capture Ag and induce tolerance or home to lymph node DCs in Lymph node Immature DCs establish T cell tolerance Mature DCs induced effector T cell fxns

Experimental System: Transgenic Mouse EYFP 11c C57BL/6 11c EYFP

Experimental System: Flourophores EYFP enhanced yellow fluorescent protein Label CD11c+ DCs ECFP enhanced cyan fluorescent protein C57/BL6 mice expressing ECFP on B-actin promoter Label DCs with different flourophore EGFP enhanced green fluorescent protein Label T and B cells

Experimental System: Two Photon Microscopy Eo hv E1 E2 E3 Light Pulsed infrared lasers Principle: Used for high resolution imaging of thick tissue sections A laser emits pulses within femtoseconds The energy from both photons excites the flourophore to a high energy state Upon relaxation, the flourophore emits light energy t

Experimental System: Two Photon Microscopy hv Pulsed infrared lasers E2.1 E1 hv E1.1 E0 E0.1 Second Harmonic Generation Two or more photons are scattered generating a single photon of exactly twice the incoming quantum energy Requires that molecules are inversion asymmetric and spatially ordered (most biologics) Used to image collagen fibers, microtubules

Experimental System: 2 photon vs Confocal http://depts.washington.edu/keck/intro.htm Accessed 1-9-07 Helmchen et al. Nat Meth. 2 (6) 2005.

Experimental System: Two Photon Microscopy Advantages of 2 photon microscopy vs confocal microscopy: Reduced damage to cells due to low energy high wavelength light Increased resolution b/c less light scattered ∞1/λ Infrared light allows for deep tissue penetration (up to 800um) Absence of absorption in out of focus planes limits photobleaching to focal plane t

Experimental Plan Created transgenic mouse with DCs expressing EYFP 2 photon microscopy to image steady-state DCs in lymph node Label T and B cells with EGFP to study interaction of DCs and lymphocytes in lymph node Study interactions of LPS activated mature ECFP expressing DCs with lymphocytes as compared with steady-state immature EYFP expressing DCs in adoptive cell transfer experiments t