Maitai hp deepsee

300μm vibratome sections from spinal columns of Clec9a^CreRosa^LSLtdTomato mice were imaged using a Zeiss 710 NLO laser scanning multiphoton microscope equipped with a 20x 1.0 NA immersion lens. A pulsed Ti:sapphire laser (Spectra Physics MaiTai HP DeepSee) tuned to 900nm was used for excitation and emission wavelengths were detected through band-pass filters of 380-485nm (second harmonic signal) and 640-690nm (tdTomato). Images were analyzed and channels adjusted using Imaris (Bitplane).

AAV1-EGFP with the synapsin I promoter (titer 1 × 10¹⁰ vg/ml, 1 μl) was injected into the retrosplenial cortex (anteroposterior, −2.0 mm from bregma and mediolateral 0.6 mm; depth, 1 mm) of 10-wk-old mice under anesthesia with 2.5% isoflurane. 2 wk later, a high-speed micro-drill was used to thin a circular area on the skull. The head of the animal was immobilized by attaching the head plate to a custom-machined stage mounted on the microscope table. Two-photon imaging was performed on a laser-scanning microscope system FV1000MPE2 (Olympus) equipped with an upright microscope (BX61WI; Olympus), a water-immersion objective lens (XLPlanN25xW; numerical aperture, 1.05), and a pulsed laser (MaiTaiHP DeepSee, Spectra Physics). EGFP was excited at 890 nm and scanned at 500–550 nm. High-magnification imaging (101.28 × 101.28 μm; 1,024 × 1,024 pixels; 1-μm Z step) of cortical layer I was performed through the thinned-skull window at 5× digital zoom. For evaluation of a single cell volume, images were analyzed by Imaris x64 software (version 7.7.2; Bitplane).

Two-photon imaging of autophagy was performed using a laser-scanning microscope system FV1000MPE2 (Olympus, Japan) equipped with an upright microscope (BX61WI, Olympus, Japan), a water-immersion objective lens (XLPlanN25xW; numerical aperture, 1.05), and a pulsed laser (MaiTaiHP DeepSee, Spectra Physics, USA). EGFP was excited at 890 nm and scanned at 500–550 nm. TAMRA-β-amyloid was excited at 1020 nm and scanned at 547–574 nm. The scanning area used for three-dimensional imaging was 100 × 100 μm (1 μm Z steps, 1,024 × 1,024 pixels, and digital zoom x 3). The LC3 labeled autophagosome in neuron of the mouse detected at cortical layer 1 were imaged through a thinned-skull window. To determine the function of autophagy, 1 μl of Aβ (TAMRA-β-amyloid 1-42: Cat;PMC-AK13-COS, 100 μM), was injected to same region as lentivirus EGFP-LC3. Images of EGFP-LC3-positive vesicles were analyzed for the number, size and signal intensity of autophagosome using IMARIS 7.2.2 (Bitplane, Switzerland).

We used a MOM-type two-photon microscope (designed by W. Denk, MPI, Heidelberg; purchased from Sutter Instruments/Science Products^{113 (link)}). The system was equipped with a mode-locked Ti:Sapphire laser tuned to 927 nm (MaiTai-HP DeepSee, Newport Spectra-Physics), two fluorescence detection channels for iGluSnFR/GCaMP6f (HQ 510/84, AHF/Chroma) and SR101 (HQ 610/75, AHF), and a water immersion objective (W Plan-Apochromat × 20 /1.0 DIC M27, Zeiss). For image acquisition, we used custom-made software (ScanM by M. Müller and T.E.) running under IGOR Pro 6.37 for Windows (Wavemetrics), taking time-lapsed 64 × 64 pixel image scans (at 9.766 Hz) or 128 × 32 pixel image scans (at 15.625 Hz). For vertical glutamate imaging in the IPL, we recorded time-lapsed 64 × 56 pixel image scans (at 11.16 Hz) using an electrically tunable lens (ETL; for details, see ref. 61 (link)).

Before siRNA injection, siRNA was labeled with Label IT siRNA Tracker Cy5 Kit without Transfection Reagent (MIR7213, Mirus, WI, USA) according to the manufacturer’s procedures. Under anesthesia with 1% isoflurane, 300 ng Cy5-labeled-siRNA (mouse PQBP1-siRNA, sc-38200, Santa Cruz Biotechnology, Dallas, TX, USA) in 1 μl volume was injected into the retrosplenial cortex (anteroposterior, −3.0 mm form bregma; lateral, 0.6 mm; depth, 0.5 mm) of Cx3cr1-GFP mouse (005582, Jackson Laboratory, Bar Harbor, MW, USA) at 21 weeks of age using in vivo jetPEI (201-10G, Polyplus-transfection, Illkirch, France). After 16 h, the skull was thinned with a high-speed micro-drill in the mouse splenial cortex. Then, the head of each mouse was immobilized by a head plate on a custom machine stage mounted on the microscope table. Two-photon imaging was performed at 5 min intervals for 30 min using a laser-scanning microscope system FV1000MPE2 (Olympus, Tokyo, Japan) equipped with an upright microscope (BX61WI, Olympus, Japan), a water-immersion objective lens (XLPlanN25xW; numerical aperture, 1.05), and a pulsed laser (MaiTaiHP DeepSee, Spectra Physics, Santa Clara, CA, USA). Then, after injecting 2.2 μg TAMRA-labeled Tau 410 (anteroposterior, −1.0 mm form bregma; lateral, 0.6 mm; depth, 0.5 mm), two-photon imaging was performed again at 5 min intervals for 30 min.