Real-time denoising enables high-sensitivity fluorescence time-lapse imaging beyond the shot-noise limit
). To transfer pretrained models, scripts were developed to convert PyTorch models to open neural network exchange models and call TensorRT builder to optimize ONNX models for a target GPU, which produced engine files that can be used by TensorRT. The construction of the engine file would eliminate dead computations, fold constants and combine operations to find an optimal schedule for model execution.Multiple animal models and fluorescence labeling methods were associated in this research.
For functional imaging of neural activity, we used transgenic mice hybridized between Rasgrf2-2A-dCre mice and Ai148 -D mice expressing Cre-dependent GCaMP6f genetically encoded calcium indicator. Craniotomy surgeries were conducted for chronic two-photon imaging as previously described. Briefly, mice were first anesthetized with 1.5% isoflurane, and a 6.0-mm-diameter craniotomy was made with a skull drill.
For time-lapse imaging of neutrophil migration, we first performed craniotomies on wild-type mice following the procedures described above. Acute brain injury caused by craniotomy induce immune responses in the brain. After surgery, neutrophils and blood vessels were simultaneously labeled by injecting 10 μg of red wheat germ agglutinin dye and 2 μg of green-fluorescence-conjugated Ly-6G/Ly-6C antibody intravenously. The two dyes were dissolved and diluted in 200 μl of 1× PBS.
For functional imaging of ATP dynamics, wild-type mice were anesthetized with intraperitoneally injected Avertin , Sigma-Aldrich). A cranial window was opened on the visual cortex, and 400–500 nl of adeno-associated virus was injected using a microsyringe pump to express GRAB) in cortical astrocytes. A 4 mm × 4 mm square coverslip was implanted to replace the skull.
. The posterior head capsule was opened using sharp forceps at room temperature in carbonated buffer solution (103 mM NaCl, 3 mM KCl, 5mM N-Tris, 10 mM trehalose, 10 mM glucose, 7 mM sucrose, 26 mM NaHCO
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