In vivo images were acquired under isoflurane anesthesia (0.5% vol isoflurane/vol O2) using a custom-built, two-photon laser-scanning microscope controlled by ScanImage written in MATLAB32 (link). Apical dendrites of layer 2/3 pyramidal neurons of the mouse barrel cortex were imaged using a 20 × /1.0 NA water immersion objective lens (Zeiss). SEP-GluA1 and dsRed2 were excited at 910 nm with a Ti:sapphire laser (Coherent) with ∼ 100 mW power delivered to the back-aperture of the objective. Green and red fluorescence signals were separated by a set of dichroic mirrors (MOM system, Sutter Instrument) and filters (ET525/50m for green channel, ET605/70m for red channel, Chroma). Images stacks were acquired at 1024 × 1024 pixels with a voxel size of 0.18 μm in x and y with a z-step of 1 μm. Representative images shown in figures were median filtered, up-scaled and contrast enhanced.
Protocol detail
In Vivo Imaging of Dendritic Spines
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Anesthesia
Cortex
Dendrites
Fluorescence
Immersion
Isoflurane
Laser scanning microscope
Lens
Mouse
Pyramidal neurons
Sapphire
Corresponding Organization :
Other organizations : Johns Hopkins Medicine, Johns Hopkins University
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Variable analysis
independent variables
- Isoflurane anesthesia (0.5% vol isoflurane/vol O2)
- Apical dendrites of layer 2/3 pyramidal neurons of the mouse barrel cortex
- Two-photon laser-scanning microscope controlled by ScanImage written in MATLAB
- 20 × /1.0 NA water immersion objective lens (Zeiss)
- SEP-GluA1 and dsRed2 excited at 910 nm with a Ti:sapphire laser (Coherent) with ~100 mW power delivered to the back-aperture of the objective
- Green and red fluorescence signals separated by a set of dichroic mirrors (MOM system, Sutter Instrument) and filters (ET525/50m for green channel, ET605/70m for red channel, Chroma)
- Images stacks acquired at 1024 × 1024 pixels with a voxel size of 0.18 μm in x and y with a z-step of 1 μm
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