After >2 weeks of recovery from surgery, GCaMP6s fluorescence was imaged using a Prairie Investigator two-photon microscopy system with a resonant galvo-scanning module (Bruker). For fluorescence excitation, we used a Ti:sapphire laser (Mai-Tai eHP, Newport) with dispersion compensation (DeepSee, Newport) tuned to λ = 920 nm. For collection, we used GaAsP photomultiplier tubes (Hamamatsu). To achieve a wide field of view, we used a 16×/0.8 numerical aperture microscope objective (Nikon) at ×1 (850 μm by 850 μm) or ×2 (425 μm by 425 μm) magnification. Laser power ranged from 40 to 75 mW at the sample depending on GCaMP6s expression levels. Photobleaching was minimal (<1%/min) for all laser powers used. A custom stainless steel light blocker (eMachineShop.com ) was mounted to the head plate and interlocked with a tube around the objective to prevent light from the visual stimulus monitor from reaching the photomultiplier tubes. During imaging experiments, the polypropylene tube supporting the mouse was suspended from the behavior platform with high-tension springs (Small Parts) to reduce movement artifacts.
0.8 numerical aperture microscope objective
Manufactured by Nikon
The 16 × 0.8 numerical aperture microscope objective is a high-performance lens designed for use in a variety of microscopy applications. It provides a magnification of 16× and a numerical aperture of 0.8, which enables it to collect a large amount of light and produce high-resolution images. This objective is suitable for a wide range of sample types and can be used in various microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Gaasp photomultiplier tube, by Hamamatsu Photonics (4 mentions)
Two photon microscopy system, by Bruker (2 mentions)
Resonant galvo scanning module, by Bruker (2 mentions)
Lsk gr08, by Thorlabs (2 mentions)
Thorimage 3, by Thorlabs (2 mentions)
Pfm450e, by Thorlabs (2 mentions)
Mai tai hp, by Spectra-Physics (2 mentions)
4 protocols using 0.8 numerical aperture microscope objective
1
Two-Photon Imaging of GCaMP6s Fluorescence
2
Two-Photon Calcium Imaging of Neural Activity
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Two-photon calcium imaging was performed on a custom-build microscope equipped with a resonant scanning module (LSK-GR08, Thorlabs), GaAsP photomultiplier tube (Hamamatsu), and a 16 × 0.8 numerical aperture microscope objective (Nikon) using ThorImage 3.1 (Thorlabs). We used a Ti-Sapphire laser (Mai Tai HP, Spectra Physics) to excite GCaMP6 at 920 nm. The FOV was 798 μm × 798 μm (512 × 512 pixels) and images were acquired at 30 Hz for single-plane recordings and at 5 Hz for multi-plane recordings. For multi-plane recordings the objective was moved between frames using a piezo objective scanner (PFM450E, Thorlabs). The piezo was allowed to settle for 35 ms in the new z position before the next frame was recorded.
3
Two-Photon Calcium Imaging of Neural Activity
4
Two-Photon Imaging of GCaMP6s Fluorescence
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For two-photon imaging experiments, GCaMP6s fluorescence was imaged using a Prairie Investigator two-photon microscopy system with a resonant galvo scanning module (Bruker). Before two-photon imaging, widefield imaging was used to identify the RSC field by alignment to surface blood vessels.
For fluorescence excitation, we used a Ti:Sapphire laser (Mai-Tai eHP, Newport) with dispersion compensation (Deep See, Newport) tuned to λ = 920 nm. For collection, we used GaAsP photomultiplier tubes (Hamamatsu). To achieve a wide field of view, we used a 16×/0.8–numerical aperture microscope objective (Nikon) at an optical zoom of 2× (425 × 425 μm field). Imaging planes at a depth of 90 to 150 μm were imaged at a frame rate of 10 Hz. Laser power ranged from 40 to 75 mW at the sample depending on GCaMP6s expression levels. Photobleaching was minimal (<1% min−1) for all laser powers used. A custom stainless-steel light blocker (eMachineShop) was mounted to the head plate and interlocked with a tube around the objective to prevent light from the visual stimulus monitor from reaching the photomultiplier tubes.
For fluorescence excitation, we used a Ti:Sapphire laser (Mai-Tai eHP, Newport) with dispersion compensation (Deep See, Newport) tuned to λ = 920 nm. For collection, we used GaAsP photomultiplier tubes (Hamamatsu). To achieve a wide field of view, we used a 16×/0.8–numerical aperture microscope objective (Nikon) at an optical zoom of 2× (425 × 425 μm field). Imaging planes at a depth of 90 to 150 μm were imaged at a frame rate of 10 Hz. Laser power ranged from 40 to 75 mW at the sample depending on GCaMP6s expression levels. Photobleaching was minimal (<1% min−1) for all laser powers used. A custom stainless-steel light blocker (eMachineShop) was mounted to the head plate and interlocked with a tube around the objective to prevent light from the visual stimulus monitor from reaching the photomultiplier tubes.
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