Photoactivation of PA-GFP was performed to reveal visual inputs to the aMe from compound eyes. The aMe was marked by tdtomato expression with Pdf-Gal4 and mSPA-GFP was pan-neuronally expressed with GMR57C10-LexA. We used Nikon A1R+ multiphoton laser scanning microscope to image and photoactivate preparations. After defining the ROI, a laser beam of 720 nm was used for activation with a dwell time of 4.8 μs per pixel for 30 times at intervals of 8 s. Three photoactivation cycles were used at a 1-min interval. After photoactivation, two-photon images with 950 nm were acquired after a 10-min wait for GFP diffusion.
A1r multiphoton laser scanning microscope
Manufactured by Nikon
The A1R+ multiphoton laser scanning microscope is a high-performance imaging system designed for advanced fluorescence microscopy applications. It features a powerful, tunable multi-photon laser that enables deep tissue imaging with reduced phototoxicity. The A1R+ provides users with high-resolution, 3D imaging capabilities to support a wide range of scientific research and analysis.
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2 protocols using a1r multiphoton laser scanning microscope
1
Photoactivation of Visual Inputs to the aMe
2
Simultaneous Calcium Imaging and Patch-Clamp
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Fluorescence was imaged on Nikon A1R+ multiphoton laser scanning microscope, which is equipped with a GaAsP type non-descanned detector (GaAsP-NDD) for high-sensitivity detection of fluorescence signals at 950-nm wavelength, 7 frames per second, 126 × 512 pixels. Patch-clamp recordings were simultaneously performed on the targeted clock neurons. ROIs were selected manually. The fluorescence change was calculated as: ΔF/F = (Ft − F0)/F0 × 100%, where Ft is the fluorescence at time point t, and F0 is the basal fluorescence. ROI fluorescence was subtracted by the background fluorescence.
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