Cranial window implantation was performed as previously described23 (link)24 (link). In brief, mice were anesthetized with an intraperitoneal injection of ketamine–xylazine (ketamine 140 mg kg−1, xylazine 10 mg kg−1). In addition, dexamethasone (6 mg kg−1) was injected intraperitoneally to prevent the development of cerebral oedema24 (link). Mice were then placed on a heating blanket and stabilized in a stereotaxic frame. Using a dental drill (Schick-Technikmaster C1; Pluraden), a round craniotomy was made and immediately covered with round coverslip (3 mm). The coverslip was sealed to the skull using dental acrylic (Cyano-Veneer fast; Schein). A small metal bar was cemented next to the coverslip to allow repositioning of the mouse during subsequent imaging sessions. After surgery, mice received subcutaneous doses of the analgesic Carprofen (7.5 mg kg−1) and the antibiotic cefotaxime (5 mg kg−1). Imaging started 21 days after surgery to allow mice to recover and cranial windows to become clear. For time-lapse imaging of the somatosensory cortex, animals were anesthetized as described above and placed into a metal holder. Movies were acquired using the Olympus FVMPE-RS system as described for spinal cord imaging.
Fvmpe rs system
Manufactured by Olympus
Sourced in Japan
The FVMPE-RS system is a high-performance multiphoton microscope designed for advanced imaging applications. It features a high-sensitivity, high-resolution optical system and supports a range of advanced imaging techniques such as multiphoton excitation, fluorescence lifetime imaging, and second harmonic generation. The system is intended to provide researchers with a powerful tool for studying complex biological samples and phenomena.
Automatically generated - may contain errors
Lab products found in correlation
Xlpln25xwmp2, by Olympus (2 mentions)
Maitai deepsee ti sapphire laser, by Spectra-Physics (2 mentions)
Insight x3, by Spectra-Physics (2 mentions)
Fluoview software, by Olympus (2 mentions)
Deepsee ti sapphire laser, by Spectra-Physics (1 mentions)
Xlpln25xsvmp2, by Olympus (1 mentions)
Gaasp photomultiplier tube, by Hamamatsu Photonics (1 mentions)
Insight ds dual, by Spectra-Physics (1 mentions)
Fv30 fgr, by Olympus (1 mentions)
Insight ds ol, by Spectra-Physics (1 mentions)
Matlab, by MathWorks (1 mentions)
8 protocols using fvmpe rs system
1
Cranial Window Implantation for In Vivo Imaging
2
In Vivo Intravital Fluorescence Imaging
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Prior to imaging, mice were anaesthetized and maintained as previously described. After induction, the head of the mouse was firmly fixed and positioned under the microscope objective using the head plate and holding bar. A fluorescent vascular tracer (70 kDa Dextran-TxRed) was injected i.v. to label blood vessels. Imaging was performed using a high NA, 25X magnification objective (XLPLN25XWMP2, Olympus) on a FVMPE-RS system (Olympus) using FluoView software. Image processing was performed with Fiji (NIH, USA).
3
In vivo Two-Photon Calcium Imaging
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The in vivo two-photon calcium imaging procedures were previously described (Huang et al., 2016 (link), 2020 (link)). In vivo two-photon calcium imaging was performed with an Olympus FVMPE-RS system with the laser tuned to the optimal excitation wavelength for YFP (920 nm) equipped with a Deepsee Ti:Sapphire laser (MaiTai DeepSee, Spectra Physics). All experiments were performed using a 25 × water immersion objective (numerical aperture 1.1) immersed in an ACSF solution and with a 1.5 × digital zoom. The average laser power on the tissue sample was 10–40 mW for imaging to minimize phototoxicity. All the calcium signals were recorded at 2 Hz with a resolution of 512 × 512 pixels. The total imaging stack of each mouse consisted of 120 frames (about 1.6 min).
The neuronal activity was monitored at 5 time points: before anesthesia (baseline, –2 h), immediately after the anesthesia and surgery (0 h), 2 h after the anesthesia and surgery when the mice started to wake up and exhibited voluntary movements (2 h), 4 h (4 h), and 6 h (6 h) after the anesthesia and surgery.
The neuronal activity was monitored at 5 time points: before anesthesia (baseline, –2 h), immediately after the anesthesia and surgery (0 h), 2 h after the anesthesia and surgery when the mice started to wake up and exhibited voluntary movements (2 h), 4 h (4 h), and 6 h (6 h) after the anesthesia and surgery.
4
Multiphoton Imaging for Neuronal Analysis
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Multiphoton images were acquired with an FVMPE-RS system (Olympus), a 25× water immersion objective lens (XLPLN25XSVMP2, Olympus), and a broadly tunable laser with a pulse width of 120 fs and a repetition rate of 80 MHz (InSight DS+ Dual, Spectra Physics) as previously described (56 ). The signals passed through emission filters of 410 to 455 nm or 495 to 540 nm (V30-FVG, Olympus) and were collected using a GaAsP photomultiplier tube (Hamamatsu Photonics). The frame acquisition rate was 1 frame/s, with two galvanometric scanning mirrors for the x and y axes. The pixel dwell time was 2 μs, and the size of the imaging fields was 512 × 512 pixels (0.994 μm per pixel for pipette imaging and 0.249 μm per pixel for in vivo imaging). To take pipette images, pipettes were filled with Milli-Q, and the images were z-stacked. For whole-cell recordings, pipettes were filled with intracellular solution containing 135 mM K-gluconate, 4.0 mM KCl, 10 mM Hepes, 0.3 mM EGTA, 10 mM Na2-phosphocreatine, 4.0 mM MgATP, 0.3 mM Na2GTP, and 0.2% biocytin.
5
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.
6
KCl-Triggered Spreading Depolarization Imaging
7
Two-Photon Microscopy Imaging Protocol
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All images were collected by an Olympus FVMPE-RS system (Olympus, Center Valley, PA) using an Olympus 25× water objective (XLPLN25XWMP2, 1.05NA). The system was equipped with two two-photon lasers: Spectra-Physics InSightX3 (680nm-1300nm, Spectra-Physics, Santa Clara, CA) and Spectra-Physics MaiTai DeepSee Ti:Sapphire laser (690nm-1040nm). There were four Photon Multiplier Tubes (PMTs) and two filter cubes (Blue/Green cube: 420-460nm/495-540nm, Red/Far Red cube: 575-630nm/645-685nm) for multi-color imaging. A galvanometer scanner was used for scanning, and all images were acquired at ~1 frame/s. PMT gains for all imaging were used between 500 and 700 airy units in the Olympus Fluoview software.
8
Multimodal Microscopy Imaging Protocol
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All images were collected by an Olympus FVMPE-RS system (Olympus, Center Valley, PA) using Olympus 25´ water objective (XLPLN25XWMP2, 1.05NA). The system was equipped with two two-photon lasers: Spectra-Physics InSightX3 (680nm-1300nm, Spectra-Physics, Santa Clara, CA) and Spectra-Physics MaiTai DeepSee Ti:Sapphire laser (690nm-1040nm). There were four Photon Multiplier Tubes (PMTs) and two filter cubes (Blue/Green cube: 420-460nm/495-540nm, red/fRed cube: 575-630nm/645-685nm) for multi-color imaging.
Galvonometer scanner was used for scanning, and all images were acquired at ~1frame/s. PMT gains for all imaging were used between 550 and 700 a.u. in the Olympus Fluoview software. All images were registered within Matlab (MathWorks) using the registration code provided by the Center for Integrated Research Computing (https://github.com/TophamLab/Registration) and were analyzed and visualized using Imaris (Bitplane) software.
Galvonometer scanner was used for scanning, and all images were acquired at ~1frame/s. PMT gains for all imaging were used between 550 and 700 a.u. in the Olympus Fluoview software. All images were registered within Matlab (MathWorks) using the registration code provided by the Center for Integrated Research Computing (https://github.com/TophamLab/Registration) and were analyzed and visualized using Imaris (Bitplane) software.
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