Mai tai deepsee laser

While under sedation, vertical flank incisions were made to externalize left kidneys for imaging^{10 (link)}. In some cases, the internal jugular vein was cannulated for intravenous infusions of dyes. Body temperature was controlled, as exteriorized kidneys were positioned inside a glass-bottom dish containing saline, which was set above a 60× water-immersion objective. Fluorescent micrographs were collected using an Olympus (Center Valley, PA) FV 1000-MPE Microscope equipped with a Spectra-Physics (Santa Clara, CA) MaiTai Deep See laser, with dispersion compensation for two-photon microscopy, tuned to 770–860 nm excitation wavelengths. The system was mounted on an Olympus IX81 inverted microscope, was also equipped with dichroic mirrors to collect blue, green, and red emissions and two external detectors for two-photon imaging.

Mice were anaesthetized and prepared for intravital microscopy as described previously [39 (link)]. Two-photon imaging was performed with a W Plan-Apochromat 20x/1,0 DIC VIS-IR objective (Zeiss) on a LSM 700 confocal laser scanning microscope (Zeiss) and a Mai Tai DeepSee laser (Spectra-Physics) tuned at 920 nm. For analysis of parasite proliferation in vivo, the emitted mKikume signal and second harmonics were split with 625 nm long pass, 495 nm long pass, and 555 nm long pass dichroic mirrors and filtered with 470/20 (second harmonics), 525/50 (mKikume green) and 600/40 (mKikume red) nm bandpass filters before collection with nondescanned detectors. For intravital analysis of cell-to-cell transmission, ECFP, EYFP and DsRed fluorescence as well as harmonics were split with 560 nm long pass, 470 nm long pass, and 520 nm long pass dichroic mirrors and filtered with 600/40 (DsRed), 470/20 (second harmonics), 506/20 (ECFP) and 543/20 (EYFP) nm bandpass filters. Typically, imaging volumes of 0.8 mm³ for automated analysis were obtained by collecting 3–4 μm spaced z stacks using the ZEN acquisition software (Zeiss). Images were color corrected using the channel arithmetics function, superimposed and analyzed using the Imaris software (Bitplane), 3D projections and slices were extracted using the Fiji software (NIH, http://rsb.info.nih.gov/ij/).

Anesthetized animals were placed on the microscope stage with the exposed intact kidney placed in a coverslip-bottomed cell culture dish bathed in warmed (37°C) isotonic saline, as previously described [38 (link), 39 (link)]. Live animal imaging was performed using an Olympus FV1000-MPE confocal/multiphoton scanner. The scanner is equipped with a Spectra Physics Mai Tai DeepSee laser and 12-bit gallium arsenide detectors. For experiments examining kidney oxidative stress, carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-DCFDA; Thermo Fisher Scientific/Invitrogen, Carlsbad, CA) was administered intravenously (7 mg/kg) 20 minutes prior to intravital imaging as previously described [35 (link)]. For experiments examining leukocyte adhesion, a fluorescein isothiocyanate- (FITC-) conjugated dextran (500 kDa; TDB Consultancy AB, Uppsala, Sweden) was injected intravenously at the time of intravital imaging to define the vascular space as previously described [39 (link)], and leukocyte adhesion to the microvascular endothelium was analyzed as previously described [40 (link)]. Quantitative analysis of acquired images was performed with Metamorph software.