Multimodal Imaging of Tissue Structure

Paraffin-embedded sections were imaged using a Zeiss LSM 780 confocal microscope with a 3 watt Chameleon Vision II (Coherent Inc, Santa Clara, CA) Ti:Sapphire oscillator used to generate 140 fs laser pulses at 820 nm with a 80 MHz repetition rate. A 40× 1.4 numerical aperture (NA) PlanApo oil immersion objective (Zeiss 420762-9900) and 1.2 NA water immersed condenser (Zeiss, Oberkochen, Germany) were used. The 410nm, second harmonic signals generated in the tissue were detected using a 400 ± 20 nm filter (Chroma Technology Corp., Bellows Falls, VT), and a 525 ± 50 nm filter (Chroma Technology Corp.) was used for two-photon excited fluorescence (TPEF) detection. TPEF was used to visualize elastin autofluroescence and a nondescanned detection (NDD) system was used for detection of SHG in the forward direction using a photomultiplier tube (LSM NDD; Zeiss) and in the reverse direction using a GaAsP detector (LSM BiG; Zeiss) to visualize collagen.^{33 (link), 34 (link)}