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.
Resonant galvo scanning module
Manufactured by Bruker
The Resonant galvo-scanning module is a core component of optical scanning systems. It utilizes high-speed galvanometer scanners to enable rapid and precise beam steering for applications such as laser scanning microscopy, holography, and industrial laser processing.
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3 protocols using resonant galvo scanning module
1
Two-Photon Imaging of GCaMP6s Fluorescence
2
Two-Photon Imaging of GCaMP6s Fluorescence
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After >2 weeks recovery from surgery, GCaMP6s fluorescence was imaged using a Prairie Investigator two-photon microscopy system with a resonant galvo-scanning module (Bruker). Prior to two-photon imaging, epifluorescence imaging was used to identify the visual area being imaged by aligning to areal maps measured with wide-field imaging.
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 NA microscope objective (Nikon) at 1× (850 × 850 μm) or 2× (425 × 425 μm) magnification. 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.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 PMTs. 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.
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 NA microscope objective (Nikon) at 1× (850 × 850 μm) or 2× (425 × 425 μm) magnification. 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.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 PMTs. 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.
3
Two-Photon Imaging of GCaMP6s Fluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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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