Hcx apo l 40 objective

Neuron voltage clamp photo-stimulation experiments were done with a LED mounted on a microscope for widefield illumination (Leica 3000B), with a nominal wavelength at 480 nm (X-Cite XLED1, Excelitas Tecnologies). We filtered the LED light with the 472/30 nm BrightLine single-band bandpass filter (Semrock). Light was triggered by pClamp (Molecular Devices). Light power was measured as 34.84 mW/mm², through a Leica HCX APO L 40× objective (air, NA=0.6). For each trace recorded, a 10 ms current injection was given (to make sure a neuron could spike), followed by a 1 ms light pulse (480nm; 34.84 mW/mm²) 5 seconds later.

GFP-fusions with trafficking sequences, opsin-GFP with trafficking sequences, and cytosolic mCherry expressed in cultured neurons were imaged with a LED mounted (X-Cite XLED1, Excelitas Technologies) on a microscope for widefield illumination (Leica 3000B), through either a Leica HCX APO L 40× objective (air, NA=0.6) or a Leica HCX APO L 20× objective (air, NA=0.5). Imaging was performed with a Hamamatsu Orca Flash 4.0 camera under identical illumination conditions throughout: a 480 nm LED using GFP-3035D filter cube (Semrock) for GFP fluorescence (34.84 mW/mm²) and a 540 nm LED with 543 nm ± 11 nm excitation filter (Semrock) for mCherry fluorescence. Images were taken with an exposure time of 300 ms.
Cultured neurons expressing CoChR-GFP, KA2-GFP or KA2(1–150)-GFP were imaged using similar parameters: fluorescence was excited with a 480 nm LED filtered by a 472/30 nm BrightLine single-band bandpass filter (Semrock) and focused on the sample through a Leica HCX APO L 20× objective (air, NA = 0.6), with a power of 25.19 mW/mm². Images were acquired with a Hamamatsu Orca Flash 4.0 with an exposure time of 300 ms.