Tcs sp8 multiphoton microscope

For multiphoton microscopy, cells were stained with Lysosensor DND-160 (Thermo Fisher Scientific) and imaged within 12 min using a Leica-TCS SP8 Multiphoton microscope set at 760 nm double photon excitation with a ×25/0.95NA HC FLUOSTAR water immersion lens. Separate channels were used to collect emitted fluorescence, corrected total cell fluorescence was calculated and a ratio between the blue and green channels generated to estimate lysosomal acidity.

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.

Silicon grids with drop-cast gold nanoparticles were mounted using Cytoseal 60 (Richard Allan Scientific Co., San Diego, CA, USA) and coverslipped. Images and emission spectra acquisition of gold nanoparticles on silicon finder grids was performed using a Leica TCS SP8 multiphoton microscope (Leica Microsystems, Wetzlar, Germany). Grid regions previously identified via SEM were imaged using a HC PL APO 63×/1.40 OIL CS2 objective (Leica Microsystems, Wetzlar, Germany). Tiling was used to image larger grid areas; then, the scanning region was reduced to gather the emission spectrum and to gather images for co-registration. Spectral data from gold–silica nanoparticles were acquired with 10 nm spectral resolution. A bleaching study was performed by drying a solution of gold–silica nanoparticles and fluorescein onto quartz slides and illuminating the field of view and then shifting the area of illumination. Gold and fluorescein intensities were recorded via specific region of interest (ROI) selection.