Lambda xl

Comparative study regarding the photo-stable characteristic of pristine dye and dye-loaded nanoparticles was conducted using xenon arc light source (Lambda XL, Sutter instrument, USA). As shown in Fig. S-3, the distance between the light source and the cuvette was fixed. Then, the fluorescence intensity from two cuvettes with a dye-only solution and colloidal NF-SIONs solution was measured at 675 nm wavelength by 1 minutes’ interval under continuous illumination. The amount of Cy 5.5 in each solution was set to be identical, based on the calculated amount of loaded dye in NF-SIONs. The initial light intensity was measured by an optical power meter, then divided by the illuminated volume at the same position. The calculated power per unit volume was 2.65 W·cm⁻³ and hence the accumulated illumination dose was calculated as below. $$\mathrm{Illumination}\mathrm{dose}[\mathrm{J}·{\mathrm{cm}}^{-3}]=\mathrm{Light}\mathrm{power}\mathrm{per}\mathrm{volume}[\mathrm{W}·{\mathrm{cm}}^{-3}]\times \mathrm{Illumination}\mathrm{time}[\mathrm{s}]$$

Worms expressing ChR2 first grew to L1-L2 stage on standard NGM plates. They were then transferred to new NGM plates either with or without (for negative control) all-trans retinal 2 days before experiments. The retinal plates were prepared by spotting each plate (60-mm diameter with 10-ml agar) with 200-µl OP50 containing 2-mM retinal (R2500, Sigma-Aldrich). In most experiments, blue light pulses (2 sec or 5 sec, 470 ± 20 nm) were generated by a Lambda XL light source (Sutter Instrument, Novato, CA, USA) with a 470 ± 20-nm excitation filter (59222, Chroma Technology Corp., Bellows Falls, VT, USA) and SmartShutter^® (Sutter Instrument). Light intensity was adjusted by applying the three standard neural density filters (ND4, ND8, ND16) of the Nikon FN1 microscope in various combinations, which resulted in eight different light intensities ranging from 0.01 to 4.38 mW/mm². The maximal light intensity was used in all experiments except those assessing the effect of light intensity-dependent activation of ASH neurons on AVA membrane potential. Light intensities were measured with an optical power meter (PM100A, Thorlabs, Newton, NJ, USA) equipped with a photodiode power sensor (S121C, Thorlabs). The on-and-off of light stimulation was controlled by NIS-Elements imaging software (version 4.51) through the SmartShutter in the Lambda XL light source.

Worms of an integrated strain expressing channelrhodopsin-2 in command interneurons under the control of Pglr-1 (ref. 16 (link)) were grown to L1-L2 stage on standard worm culture plates, and then transferred to new plates either with or without (for negative control) all-trans retinal two days before the experiment. The retinal plates were prepared by spotting each plate (60-mm diameter with 10 ml agar) with 200 μl OP50 E. coli containing 2 mM retinal (R2500, Sigma-Aldrich). Photostimulation was applied through a 40X water immersion objective in 2-s pulses at 30-s intervals using a light source (Lambda XL with SmartShutter, Sutter Instrument) and a 470±20 nm excitation filter (59222, Chroma Technology Corp.). The measured light intensity at the specimen position was 6.7 mW mm⁻², which was sufficient to cause maximal evoked peak responses16 (link).

The status of cell health and morphology was viewed using a phase-contrast microscope (IX73, Olympus) and images were acquired with a Retiga R1 camera (QImaging) and the ImageJ plugin Micro-Manager (UCSF). Cell viability was determined with a fluorescence-based reporter dye kit (LIVE-DEAD^TM Cell Imaging Kit, ThermoFisher, Cat# R37601). After treatment, cells were washed thrice with PBS, then incubated in HBSS (Gibco, Cat# 14025-076) containing 1 µM Calcein AM and 2 µM ethidium homodimer at 37 °C for 45 min. Images were taken using an inverted microscope (IX73, Olympus) equipped with fluorescence light filters (Lambda XL, Sutter Instrument) and a Retiga R1 camera. Cell viability was determined by manually counting the number and calculating the ratio of ethidium-homodimer-stained cells to total cells (i.e., those stained with either Calcein AM or ethidium homodimer) in each image. The neurite number and status (primary, secondary, or tertiary) were recorded using the ImageJ plugin NeuronJ (NIH).

Cells on coverslips were loaded with 5 μM Fluo-4 AM (Beyotime Bio-Tech Co., Ltd.) for 40 min at 37^oC in the original culture room. The cells were then washed three times with an incubation buffer containing (in mM) 140 NaCl, 5 KCl, 2 MgCl₂, 10 HEPES, 10 glucose, and 2 CaCl₂ (pH 7.4). Cells were incubated in incubation buffer for 30 min at 37^oC to allow deesterification of intracellular AM esters. Calcium imaging was performed on an inverted epifluorescence microscope (Olympus IX 73) equipped with a complete illumination system (Lambda XL, Sutter Instruments). Fluorescent images were acquired using a cool CCD camera (CoolSNAP ES2, Teledyne Photometrics) controlled by Micro-Manager 1.4 (Vale lab, UCSF) using a public 1394 digital camera driver (Carnegie Mellon University). Images at excitation of 448–492 nm and emission of 512–630 nm were taken in a continuous time-based mode. After baseline images were taken, 1,4-dioxane was added. High KCl (60 mM) was applied in the end of each experiment to ascertain neuronal viability. More than 90% neurons were responsive to 60 mM KCl stimulation and were included in the analysis.