Hcx pl apo l 20x 1.0 w water immersion lens

A custom 3D printed, 2 chamber specimen-holder (Figure S1B) was designed to allow for cleared tissue samples to be imaged using a Leica HCX PL APO L 20x/1.0 W water immersion lens equipped with a motorized correction collar. Briefly, eyes are immersed in imaging oil (Cargille, #16212) and immobilized in a lower, sealed specimen chamber that is capped by a #1, 22 x 22 mm coverslip (VWR, #16004-094) which separates the lower and upper chambers. An upper chamber is added that is filled with distilled water for use with the Leica HCX PL APO L 20x/1.0 W water immersion lens. Whole eyes were imaged on a dual beam Leica TCS SP8 multiphoton microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 1300nm Chameleon Discovery laser (Coherent, Santa Clara CA, USA). For imaging of GFP, the laser was tuned to 960nm. TdTomato was excited using a fixed-beam laser at 1040nm. Power output for GFP and tdTomato channels was maintained at 45mW. Topro-3 iodide was excited at 1300 nm at 300 mW. Emitted light from all fluorophores was captured using an internal HyD SP GaAsP hybrid detector gated to a range of 500-525 nm for GFP, 550-590 for tdTomato and 630-680 nm for Topro-3. Z-stack images were acquired with 2x accumulation, 2x averaging, 0.70 μm z-resolution and a minimum of 960x960 xy resolution.