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

2. 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

3. 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

4. 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

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

6. 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

7. Experimental System: Two Photon MicroscopyEo 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

8. Experimental System: Two Photon Microscopyhv
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

9. Experimental System: 2 photon vs Confocal
Accessed
Helmchen et al. Nat Meth. 2 (6) 2005.

10. 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

11. 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