VSD imaging was performed as described previously2 (link). 1–2% isoflurane was used to anesthetize the animals. After being placed in a stereotaxic apparatus, heating pad was used to maintain animal’s body temperature throughout the length of anesthesia. Each animal’s scalp was disinfected with betadine followed by isopropyl alcohol. An incision was made. A round 5–8 mm craniotomy was performed over somatosensory cortex. After dura removal, 1 mg/ml Rh1691 in phosphate-buffered saline (PBS) (Optical Imaging) was applied directly to the brain for 1 hour. Then the cortex was washed with PBS to remove unbound dye. A 5–8 mm glass coverslip was attached to the surrounding skull using dental cement and Krazy glue.
Wide field VSD imaging was carried out using a 2X long working distance objective (NA = 0.14) on an upright microscope (BX51, Olympus). 120 W mercury arc lamp was used to excite the VSD with 585/20 nm light. Hamamatsu ORCA-ER digital CCD camera was used to collect images with 5 ms exposure every 50 ms. Image sequences were acquired in 50 s epochs, up to 10 epochs/condition.
Wide field VSD imaging was carried out using a 2X long working distance objective (NA = 0.14) on an upright microscope (BX51, Olympus). 120 W mercury arc lamp was used to excite the VSD with 585/20 nm light. Hamamatsu ORCA-ER digital CCD camera was used to collect images with 5 ms exposure every 50 ms. Image sequences were acquired in 50 s epochs, up to 10 epochs/condition.
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