Fmt 2000 system

Mice were anesthetized with ketamine-xylazine anesthesia for in vivo imaging with IVIS Lumina II (PerkinElmer; 120s acquisition, F/stop = 1, Binning = 8) instrument and Living Image^® software (PerkinElmer). Luminol sodium salt (5-amino-2,3-dihydro-1,4-phthalazine-dione; 150 mg/kg) injection was given i. p. in sterile PBS solution for MPO imaging. MPO from neutrophil granulocytes produces reactive oxygen species which interact with luminol and result in luminescent signals which we measured 10 min after administration. Luminescence was expressed as total radiance (total photon flux/s) in identical Regions of Interests (ROIs) around the joints.
Inflammatory vascular leakage was evaluated by fluorescence imaging with the Fluorescent Molecular Tomography (FMT) 2000 system (PerkinElmer Ltd.) using the 680 nm laser of the equipment. A micellar formulation of the fluorescent IR-676 dye (Spectrum-Info) dissolved in a 5 w/v% aqueous solution of Kolliphor HS 15 (polyethylene-glycol-15-hydroxystearate; Sigma-Aldrich) was given i. v. in a 0.5 mg/kg dose under ketamine-xylazine anesthesia. The measurement was carried out 20 min afterward dye administration with fluorescence expressed as the calculated amount of fluorophore (pmol) (Botz et al., 2015 (link)) in identical Regions of Interests (ROIs) around the ankle joints.

The near-infrared fluorescence imaging probe ProSense 680 (PerkinElmer, Inc., Waltham, MA) was used for the visualization of protease activities including cathepsin B, L, S and plasmin. In the mouse IR model, 5 nmol of ProSense 680 (Ex/Em = 680/700 nm) was intravenously administered 48 h after reperfusion. Mice were anesthetized 72 h later with isoflurane (1.5%) inhalation and scanned with the FMT-2000 system (PerkinElmer, Inc., Waltham, MA). Next, the mice were euthanized, and the brains were harvested. The excised brains were cut into sequential cross sections of 2.0 mm thickness, stained with TTC and imaged by FRI by using the planar imaging capability of FMT-2000.