This study was performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and the University of Maryland, School of Medicine, Animal Care and Use Committee. One male C57BL6J mouse was used for in vivo testing. One microliter of green fluorescent beads (505/515, 0.1 μm diameter, Thermofisher, #F8803, 25 times diluted with phosphate buffer solution (PBS) was injected into the cerebral cortex using either pulled glass pipettes (right side of the hemisphere) connected to a Narishige micromanipulator (#MO10) or Hamilton syringe needle 33 Gauge (left side of the brain) connected to a motorized pump (KD Scientific, #78–8130). The Bregma coordinates for the three injection sites for each hemisphere were −1, 0, and 1 mm. Midline and depth were the same among the sites: 2 and 0.8 mm, respectively. The injection speed was 0.5 μL/min. The animal was perfused with 4% paraformaldehyde 11 days after the injection of fluorescent beads and dehydrated in 30% sucrose in PBS before being cryosectioned (Cryostar NX50, Epredia) at 40 μm for observation under fluorescence microscopy (Leica).
Green fluorescent beads
Manufactured by Thermo Fisher Scientific
Green fluorescent beads are laboratory-grade particles that emit green light when exposed to a specific wavelength of light. They are designed for use in various scientific applications, such as cell imaging, flow cytometry, and bead-based assays. The beads have a consistent size and fluorescent intensity, making them a reliable tool for calibration and standardization purposes.
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4 protocols using green fluorescent beads
1
Fluorescent Bead Injection in Mouse Brain
2
Imaging Neuronal Activity in Zebrafish Larvae
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Two Pan-neuronal nuclear localized GCaMP6s Tg(HuC:H2B:GCaMP6s) and two pan-neuronal soma localized GCaMP7f Tg(HuC:somaGCaMP7f) [31 (link)] zebrafish larvae were imaged at 4–6 days post fertilization. Additionally, two NLS GCaMP6s fish of unknown age. The transgenic larvae were kept at and paralyzed in standard fish water containing 0.25 mg/ml of pancuronium bromide (Sigma-Aldrich) for 2 min before imaging to reduce motion. The paralyzed larvae were then embedded in agar with 0.5% agarose (SeaKem GTG) and 1% low-melting point agarose (Sigma-Aldrich) in Petri dishes.
Additionally, a beads dataset was created by imaging green fluorescent beads (ThermoFisher) randomly distributed in 1% low-melting-point agarose (Sigma-Aldrich). The stock beads were serially diluted using melted agarose to , , , of the original concentration.
Each fish was imaged for 1000 frames at 10Hz. Neural activity images were extracted using the SLNet [32 ], as seen inFig. 1(a) . Later, the resulting images were 3D reconstructed with the RL algorithm for 100 iterations, which takes roughly per frame.
Additionally, a beads dataset was created by imaging green fluorescent beads (ThermoFisher) randomly distributed in 1% low-melting-point agarose (Sigma-Aldrich). The stock beads were serially diluted using melted agarose to , , , of the original concentration.
Each fish was imaged for 1000 frames at 10Hz. Neural activity images were extracted using the SLNet [32 ], as seen in
3
Characterizing Microscope Resolution
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Green fluorescent beads (diameter: 170 nm, Thermofisher) were dried on a coverslip and then mounted to a slide. 5 × 5 μm2 images were acquired on the respective microscopes under comparable imaging conditions. 5-μm-thick z-stacks were acquired with 0.2 μm steps and the section in focus was selected to determine xy resolution. In-focus beads were imaged in xz to determine axial resolution. Full-width at half-maximum (FWHM) measurements were done using the FWHM plug-in for FIJI.
4
Fluorescent Bead Injection in Mouse Brain
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This study was performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and the University of Maryland, School of Medicine, Animal Care and Use Committee. One male C57BL6J mouse was used for in vivo testing. One microliter of green fluorescent beads (505/515, 0.1 μm diameter, Thermofisher, #F8803, 25 times diluted with phosphate buffer solution (PBS) was injected into the cerebral cortex using either pulled glass pipettes (right side of the hemisphere) connected to a Narishige micromanipulator (#MO10) or Hamilton syringe needle 33 Gauge (left side of the brain) connected to a motorized pump (KD Scientific, #78–8130). The Bregma coordinates for the three injection sites for each hemisphere were −1, 0, and 1 mm. Midline and depth were the same among the sites: 2 and 0.8 mm, respectively. The injection speed was 0.5 μL/min. The animal was perfused with 4% paraformaldehyde 11 days after the injection of fluorescent beads and dehydrated in 30% sucrose in PBS before being cryosectioned (Cryostar NX50, Epredia) at 40 μm for observation under fluorescence microscopy (Leica).
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