The role of microglia in Aβ degradation was further examined in vivo. We stereotaxically injected 2 μl of 100 µM Rho-Aβ into the lateral ventricles of CX3CR1-GFP+/− mice. GFP-positive microglia and rhodamine (Rho)-labeled plaques were imaged through a thinned skull preparation60 (link). The transgenic mice were anesthetized with 20% urethane filtered with a 0.22 µm filter (Millipore), and the skull was exposed with a midline scalp incision. An ~1-mm-diameter region of the skull over the somatosensory cortex was thinned with a high-speed drill and a microsurgical blade to a final thickness of ~40 μm. To reduce respiration-induced movement artifacts, the skull was glued to a stainless steel plate. A mode-locked laser (MaiTai HP-OL/Insight DS-OL, Spectra-Physics) was used for two-photon excitation and set to 920 nm for the imaging of GFP, 1120 nm for the imaging of Rho and 835 nm for the imaging of Methoxy-X04 using an in vivo two-photon imaging system (Olympus FVMPE-RS). Images were obtained by using a 1.05 numerical aperture, 25× water-immersion objectives and 2.0× digital zoom. The stack was typically obtained 150 μm below the pial surface61 (link).
Insight ds ol
Manufactured by Spectra-Physics
Sourced in United States
The InSight DS-OL is a high-performance optical laser diode module designed for scientific and industrial applications. It provides a stable and precise optical output with a narrow linewidth and high beam quality. The core function of the InSight DS-OL is to generate a coherent, monochromatic light source for use in various experimental and measurement setups.
Automatically generated - may contain errors
Lab products found in correlation
Fvmpe rs, by Olympus (2 mentions)
Xlpln25xwmp2, by Olympus (2 mentions)
Xl plan n 25 1.05 wmp 0 0 23 fn 18 dipping objective, by Olympus (2 mentions)
Ehpds ol, by Spectra-Physics (2 mentions)
Fluoview fvmpe rs, by Olympus (2 mentions)
Hp ol, by Spectra-Physics (1 mentions)
0.22 m filter, by Merck Group (1 mentions)
Fv30 fgr, by Olympus (1 mentions)
Fvmpe rs system, by Olympus (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Alexa fluor 568 hydrazide, by Thermo Fisher Scientific (1 mentions)
P 1000, by Sutter Instruments (1 mentions)
Fluoview fvmpe rs two photon laser scanning microscope, by Olympus (1 mentions)
Maitai hp ds ol, by Spectra-Physics (1 mentions)
Pv820, by World Precision Instruments (1 mentions)
6 protocols using insight ds ol
1
In Vivo Imaging of Microglial Aβ Clearance
2
Optogenetic Control of Nanovesicle Release
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Au-nV and CNiFER cells were imaged with a multi-photon laser scanning microscope (FVMPE-RS, Olympus). FVMPE-RS was accompanied with stimulation laser (MaiTai HP DeepSee-OL, Spectra-Physics, 100 fs pulse width) and main scanner laser (Insight DS+ -OL, Spectra Physics, 120 fs pulse width) with a repetition rate of 80 MHz. Real-time imaging of SST2 CNiFER cells was realized by detecting the fluorescence of the genetically-encoded FRET-based Ca 2+ sensor, Twitch 2B. The fluorescence emission in two channels (CFP 460-500 nm and YFP 520-560 nm) was collected by the 25x objective (XLPLN25XWMP2, Olympus, NA 1.05) when excited at 900 nm (less than 15 mW after objective). Atto 647N-labeled Au-nV (Au-Atto-nV) was detected by recording the fluorescence emission at 575-645 nm when excited at 1100 nm. Photo-stimulation on the nanovesicles was performed at 720 nm with a series of laser power (25, 50, 75, 100, 125 mW) and tornado scans (60 µm, 5, 10, 20, 40, 80 scans) to characterize the power-and scans number-dependent release of SST. Here tornado scan refers to scan setting for fast photostimulation and allows rapid scanning of a circular area. To measure the transmission of SST, 40 tornado scans (duration: 2.6 s) at 100 mW were stimulated on the core SST2 CNiFERs implants while monitoring the FRET changes on both the core and satellite implants at 0.8 s per frame (typically 400 frames).
3
Cranial Window Preparation for Multiphoton Imaging
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Cranial windows were prepared as we previously described (48 (link), 49 (link)). Mice were anesthetized with 1 to 1.5% isoflurane in 30% oxygen and 70% nitrous oxide. Body temperature was maintained at 37 ± 0.5 °C during surgery. After fixation in a stereotaxic head holder, a craniotomy (5 mm diameter) was created above the right somatosensory cortex (centered 2.5 mm lateral and 2.5 mm posterior to the bregma) using a high-speed micro drill. The window was closed with a sterile cover glass. For multiphoton imaging, Olympus FluoView FVMPE-RS upright multiphoton laser-scanning system with an Olympus XL Plan N 25 ×/1.05 WMP ∞/0–0.23/FN/18 dipping objective was used. Multiphoton excitation was performed using MAITAI eHPDS-OL and Spectra-Physics InSight DS-OL lasers (Mai Tai, Spectra-Physics). Emitted fluorescence was detected through 495 to 540 nm and 575 to 645 nm bandpass filters.
4
Imaging Neuronal Activity in Awake Mice
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Three mice were head-restrained under the objective 4–13 days after the implantation of the cranial windows and performed the same motor task during TPLSM imaging as was used during 8K-SDCLM imaging. Two-photon images were acquired using an FVMPE-RS system (Olympus, Tokyo, Japan) with a broadly tunable ultrafast laser (InSight DS-OL; Spectra Physics, CA, USA) tuned to 940 nm. The back aperture of the objective (XLPLN25XWMP2; back aperture diameter, 15.1 mm; numerical aperture [NA], 1.05; Olympus) was underfilled with the diameter-shortened (7.2 mm) laser beam to reduce the effective excitation NA of the objective14 (link). The dimensions of the FOV were 512 × 512 pixels (127.2 × 127.2 µm) for all imaging fields. The imaging fields were at 40–60 µm depth below the cortical surface. The laser power was adjusted to maintain a relatively constant fluorescent intensity from the axonal boutons (5.94–11.9 mW). A series of 10,800 continuous images was acquired at 30 Hz. A 570 nm dichroic mirror (FV30-FGR; Olympus) and a bandpass filter at 495–540 nm were used. For XYZ imaging (Fig. 5i ), three planes with an interval of 8 µm in the same horizontal field were sequentially imaged in three mice. For each plane, the dimensions of the FOV were 512 × 256 pixels (84.9 × 42.4 µm) and a series of 1860 or 2230 images was acquired at 6.2 Hz.
5
Measuring Cortical Vascular Permeability
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Cranial windows were prepared as we previously described.31,32 (link) Briefly, mice were anesthetized with 1% to 1.5% isoflurane in 30% oxygen and 70% nitrous oxide and kept on a heating plate (37°C ± 0.5°C). After fixation in a custom-made head holder, a window 2 mm in diameter was made in the parietal bone (centered 2.5 mm lateral and 1 mm posterior to the bregma) using a high-speed micro drill (Stoelting). A sterile 5 × 5 mm cover glass (World Precision Instruments) was placed above the window and fixed with dental cement. For in vivo imaging, an Olympus FluoView FVMPE-RS upright multiphoton laser-scanning system mounted on an Olympus XL Plan N 25×/1.05 WMP ∞/0-0.23/FN/18 dipping objective was used. Two-photon excitation was performed with Mai Tai eHPDS-OL (Santa Clara, CA) and Spectra Physics InSight DS-OL lasers (Santa Clara, CA). Emitted fluorescence was detected through a 495- to 540-nm band pass filter. To analyze cortical cerebrovascular permeability, FITC-dextran (molecular weight, 40 KDa; Sigma-Aldrich; 0.1 mL of 10 mg/mL) was injected IV, and time lapse imaging of FITC-dextran was acquired every 3 minutes for 30 minutes. The fluorescence of randomly chosen 20 × 20 μm2 regions of interest within the vessel and corresponding areas within the perivascular brain parenchyma was recorded as described.16 (link)
6
Two-Photon Imaging of ATP Signaling in Acute Brain Slices
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Three weeks after in vivo microinjection of AAV9, acute slices were prepared according to the procedure described in the Supplementary material . The slices were imaged at 32–34°C at a depth of 50–100 μm with a Fluoview FVMPE-RS two-photon laser scanning microscope (Olympus) equipped with a Maitai HP DS-OL (Spectra-Physics) tuned to 920 nm (laser power ∼1.65 mW) and an InSight DS-OL (Spectra-Physics) tuned to 1100 nm to image ATP and Alexa Fluor 568 Hydrazide (Invitrogen), respectively, through a 25× water-immersion objective (NA 1.05; Olympus). Images were acquired with a rectangular FOV of 120 × 82 μm with 226 × 156 pixels. ATP (1 mM) was locally administered using glass capillaries (1B150-4, World Precision Instruments) and a Pneumatic Pico Pump (PV 820, World Precision Instruments). The glass capillaries were pulled using a micropipette puller (P-1000, Sutter Instruments) with the following parameters: heat 590, pull 0, vel 40 and time 200. Imaging data analyses were performed using FIJI ImageJ software. Regions of interest (ROIs) were selected near the point where ATP was locally administered, which was encompassed by spreading ATP fluorescence signals, and mean intensity values were obtained from each ROI and converted into dF/F values using Excel. The fluorescence dynamics of ATP were analysed using OriginPro.
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