Mai tai deepsee hp ti sapphire laser

OCs from P2 Rac1-fluorescence resonance energy transfer (FRET) biosensor TG mice [30 (link)] were dissected in Leibovitz’s L-15 medium (Invitrogen), attached to 3.5-mm Cell-Tak coated dishes (150 µg/µL; BD Biosciences) and maintained in Dulbecco’s modified Eagle medium/F-12 supplemented with 10% fetal bovine serum. FRET imaging under a two-photon excitation microscope was performed as previously described [28 (link)]. Samples were maintained in an incubation chamber (Tokai Hit, Nagoya, Japan) and imaged using a BX61WI/FV1000 upright microscope equipped with a × 60 water-immersion objective (LUMPlanFLN; Olympus, Tokyo, Japan) connected to a Mai Tai DeepSee HP Ti:sapphire laser (Spectra Physics, Mountain View, CA, USA). FRET/CFP images were acquired and analyzed using MetaMorph (Universal Imaging, West Chester, PA, USA) and Imaris software (Bitplane AG, Zürich, Switzerland) and represented using the intensity-modulated display mode, in which eight colors from red to blue are used to represent the FRET/CFP ratio.

Two-photon imaging was performed using a resonant scanner B scope (Thorlabs) with a Mai Tai DeepSee HP Ti:Sapphire laser (Spectra Physics) or a Chameleon Ultra II laser (Coherent) attached to a galvo confocal scanning system (Fluoview 300, Olympus). A water-immersion 20× objective (1.0 NA, Olympus) was used on both systems. Intravascular fluorescein isothiocyanate–dextran (FITC–dextran, 2,000 kDa) and red microspheres were excited at an 820 nm wavelength. Images were acquired at 30 Hz (ThorSync software) simultaneously with physiological recordings (3 kHz, ThorSync software). Prior to infusion of the microspheres, a volumetric image of the vascular topology was acquired. Segments of the MCA were identified as large caliber surface vessels (50–60 µm diameter) that started at the anterolateral aspect of the window and traveled towards the midline. Direction of blood flow was confirmed before and after imaging using line scans.