GCaMP6f or iGluSnFR fluorescence was recorded by a 2-photon laser scanning microscope (model “Ultima”, Prairie Technologies). Images were recorded from cortical layers 2/3 with a model “XLPLN 25 × WMP” 1.05NA, water-immersion objective (Olympus), using 900–910 nm laser pulses for excitation with an average power of 6–14 mW, as measured by LaserCheck (Coherent). The laser was a model “Mai Tai DeepSee” (Spectra Physics). Time-series and electrophysiological recordings were triggered by pCLAMP 10.4 (Molecular Devices, LLC) to enable precise timing of the DC shifts and corresponding images. Most time-series were performed with frame rates of approximately 4 Hz, but a subset of experiments was performed with 1 or 6 Hz acquisition rates.
Xlpln 25 wmp
Manufactured by Olympus
The XLPLN 25 × WMP is a microscope objective lens from Olympus designed for use in laboratory and research applications. It provides a magnification of 25× and a working distance of WMP, allowing for detailed observation and analysis of samples. The core function of this product is to enable high-quality imaging and visualization of specimens under a microscope.
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2 protocols using xlpln 25 wmp
1
Cortical Fluorescence Imaging with 2-Photon
2
In Vivo Imaging of Mouse Ear Vasculature
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ZO-1 Venus mice were anesthetized with isoflurane (AbbVie, North Chicago, IL) and oxygen and air; the isoflurane concentration was initially set at 4% and gradually lowered to 1.2% in a constant oxygen flow (0.15 L/min). To prevent movement, the dorsal side of the mouse ear was fixed to the table with double-sided adhesive tape. The two-photon microscope is a custom-made upright microscope (BX61WI, Olympus, Japan) attached to a mode-locked titanium-sapphire laser system (Chameleon Vision II, Coherent, Santa Clara, CA) that achieves a 950 nm laser with a 140-fs pulse width and an 80-MHz repetition rate (Morikawa et al., 2012 (link)). Images (512 × 512 pixels) were acquired by z-stack scanning at 0.6 μm intervals with a 25× objective lens (XLPLN25 × WMP; NA 1.05, Olympus) and an Olympus FV1000 scanning unit using Fluoview software (FV10-ASW, Olympus). Emitted fluorescence was detected using an external photomultiplier tube (R3896; Hamamatsu Photonics, Japan) after reflection via a dichroic mirror (580 nm cut-off) and passing through an emission filter (500–550 nm). Acquired images were processed using Imaris software (Bitplane).
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