Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

1. Au NRs Preparation

Note: Au NRs can be synthesized by a number of recipes¹⁶, or purchased from commercial vendors.

1. Measure the initial optical density (OD) of the Au NR solution via UV-Vis spectroscopy, by recording the absorption values from 300 nm to 1,000 nm with a resolution of 0.5-2 nm. Vary the volume of the solution to be used with the available cuvette.
2. Evaluate the initial NP molar concentration with a suitable technique¹⁷ (e.g. UV-Vis spectroscopy, single particle inductively coupled plasma mass spectrometry, transmission electron microscopy) or use the concentration values provided by the vendor.
3. Prepare a 5 ml stock solution by diluting the initial Au NR sample to reach an OD = 1. For the best repeatability, keep the OD constant for all of the tested samples. Follow the commercial vendor’s protocol for the composition of the diluting solvent. If unsure, use deionized water.
4. Centrifuge 1 ml of the Au NR solution twice for 15 min at 7,800 x g to remove any chemical excess from the solution. Centrifugation cycles can vary in force and time (e.g. 20 min at 5,600 × g)¹⁸.
5. Remove the supernatants and re-suspend the NRs in deionized water. As re-suspended particles might form aggregates in solution, prepare them daily to have the best results. Alternatively, store in a fridge for no longer than 1 week. Do not freeze.
6. Before using under cell culture conditions, sonicate the Au NR solution for 5 min and then sterilize with UV light for 30 min (UV radiation intensity not less than 400 mW∙m^-2 at 254 nm).

2. NG108-15 Neuronal Cell Line Culture and Differentiation

1. For the cell culture medium, prepare 500 ml of sterile Dulbecco's modified Eagle medium (DMEM) containing 10% (w/v) fetal calf serum (FCS), 1% (w/v) L-glutamine, 1% (w/v) penicillin/streptomycin and 0.5% (w/v) amphotericin B.
   Note: Supplements can be aliquoted, stored at -20 °C and added to the media on the day required. Cell culture medium can be refrigerated in a sterile condition for a maximum of 1 month.
2. For the cell differentiation medium, prepare 50 ml of sterile DMEM containing 1% (w/v) L-glutamine, 1% (w/v) penicillin/streptomycin and 0.5% (w/v) amphotericin B.
3. Grow NG108-15 neuronal cells in 10 ml of cell culture medium in T75 flasks made of polystyrene in an incubator with humidified atmosphere (5% CO₂ at 37 °C). Normally, seed 1.5-2 × 10⁵ cells in each flask to be ready in 3-4 days. Change cell culture medium every two days.
   Note: To prevent genetic drifts or variation, do not use cells older than passage 21 for experiments.
4. When 70-80% confluent in culture, change the medium with warm fresh cell culture medium. Mechanically detach the cells by gently knocking the bottom of the confluent flask. Do not use trypsin.
5. Centrifuge the cell suspension for 5 min at 600 x g and re-suspend the cell pellet in 2 ml of warm cell differentiation medium.
6. Seed 2 × 10⁴ cells/cm² in a tissue culture polystyrene 96 well plate with 200 µl of cell differentiation medium. Incubate the experiment for 1 day at 5% CO₂/37 °C.
7. Add between 3.2 × 10⁹-4.2 ×1 0¹⁰ particles/ml of Au NR solution on day 2 and incubate it for additional 24 hr. Do not add the particles for the control experiments.
   Note: As an alternative control, Au NPs with a well-differentiated peak absorption wavelength can be used for comparison purposes¹⁴. For a more consistent cell behavior, keep the cell density constant and do not modify the well surface prior to cell seeding.

3. Neurite Outgrowth Enhancement

1. Couple the laser with a single mode optical fiber (numerical aperture = 0.13) and terminate it with a fiber connector (FC connectors are convenient and commonly available). Measure the output laser power with a standard power meter. To obtain the most effective results, match the peak wavelength of the laser to the plasmon resonance peak of the NRs.
2. On day 3 following NR incubation, fix the FC connector to the well. Irradiate samples and controls at RT for 1 min in continuous wave, for different laser powers. Allow the culture to proceed for 3 additional days at 5% CO₂/37 °C. Repeat the laser irradiation for a minimum of 3 independent measurements.
   Note: Different irradiation times and pulse frequencies may be selected, depending on the application.
3. Characterize the laser in terms of beam diameter and laser irradiance (W∙cm^-2).
   1. For a standard single mode fiber, use NA = n∙sinθ, where n is the refractive index of the medium in use and θ is the half-angle of the cone of light exiting the fiber (see Figure 1A). From trigonometry, r = tanθ∙d, where r is the beam radius and d is the distance between the fiber and the sample. The FC connector matches the diameter of the well, therefore illuminating the sample at a distance d = 2.70 ± 0.20 mm. Light exits the fiber in water (n = 1.33), giving r = tan(sin-1(NA/n))∙d.
   2. Using the latter equation, calculate the laser beam radius, the corresponding beam area and the average laser irradiance (laser power divided by beam area) at the target (see the example graph in Figure 1B). These values represent the average irradiance over the illuminated area.
   3. Evaluate the errors for the measurements with the general theory of error propagation.
      Note: The distance between the FC connector and the sample can be measured by photographs of the experimental arrangement and post-processing of the image using appropriate software (e.g. ImageJ).
4. At day 5, remove cell differentiation medium from the experiments and fix the samples with 3.7% (v/v) formaldehyde solution for 10 min, then permeabilize the cells with 0.1% (v/v) Triton X-100 for 20 min.
5. Add 3% (w/v) bovine serum albumin (BSA) to the samples for 60 min to block the unreacted protein binding sites. Label the samples for anti-βIII-tubulin overnight (5 µg/ml in PBS supplemented with 1% of BSA) at 4 °C.
6. Incubate the cells for 90 min in the dark with an appropriate secondary antibody (e.g. TRITC-conjugated anti-mouse IgG antibody) using a concentration of 0.4-2 µg/ml in 1% BSA in PBS. Label the cell nuclei with DAPI (0.1 µg/ml in de-ionized water) for 10 min.
   Note: Antibody conjugation and concentration might vary according to the company protocol. Wash samples with PBS twice for 5 min after each staining stage.
   
7. Image samples by epifluorescence or confocal microscopy using at least a 20× objective. Choose the microscope filters accordingly to the secondary antibody. Select a DAPI filter (λ_EX = 358 nm; λ_EM = 488 nm) to visualize the cell nuclei.
8. Analyze the pictures by assessing: i) the maximum neurite length (record the length from the tip of the neurite to the beginning of the cell body), ii) the number of neurites per neuronal cell (sum up all of the neurites per cell) and iii) the percentage of cells with neurites (divide the total number of cells expressing βIII-tubulin by the total number of cells with a positively stained nucleus)¹⁹.

4. Laser-induced Intracellular Ca²⁺ Imaging

1. Prepare a 20% (w/v) stock solution of Pluronic F-127 by dissolving 2 g of solute in 10 ml of anhydrous dimethyl sulfoxide (DMSO). Heat the solution of Pluronic F-127 at 40 °C for about 20 min to increase the solubility. Prepare it in advance if stored at RT.
2. On day 3 following NR incubation, prepare a balanced salt solution (BSS; 135 mM NaCl, 4.5 mM KCl, 1.5 mM CaCl₂ , 0.5 mM MgCl₂ , 5.6 mM glucose, and 10 mM HEPES, pH 7.4) and supplemented it with 5 µM of Fluo-4 AM in DMSO and 0.1% (w/v) of Pluronic F-127 solution.
   Note: BSS solution can be prepared in advance and refrigerated for a maximum of 1 month. Add the supplements (Fluo-4 AM and Pluronic F-127) on the day of the experiment.
3. Remove cell differentiation medium and replace it with the supplemented BSS solution. To obtain the best results with an inverted confocal microscope, incubate the cells in a micro-slide well.
4. Load NG108-15 cells with Fluo-4 AM for 30 min in the dark at 5% CO₂/37 °C. Following the incubation time, wash samples twice with BSS to remove any extracellular unloaded fluorophore.
5. Couple the laser with a single mode optical fiber and cleave the tip using standard techniques. Observe the resulting tip under an optical microscope, to ensure a high-quality surface (i.e. the tip should be perpendicular to the fiber axis and flat upon microscopy inspection).
   1. Measure the output laser power with a standard power meter. Insert the light delivery fiber into an optical fiber holder and affix it to a micropositioner. Refer to Brown et al.²⁰ for more details on optical fiber preparation and positioning.
      Caution: Follow the general rules for laser safety during the measurement of the laser power (e.g. do not look directly into laser beam, wear laser safety goggles during handling, prevent stray light exposure to other lab users).
6. Connect an oscilloscope to the portable laser to monitor the optical modulation. Use a binary signal with variable frequencies (0.5-2 Hz) and pulse lengths (20-100 msec). Connect the laser using the modulation signal as transistor-transistor logic (TTL) input for the microscope, following the setup shown in Paviolo et al.¹³.
   1. Place the cells under an inverted confocal microscope and position the light delivery fiber 250 ± 50 µm away from the target cell in transmission illumination mode. At this distance, use the equations presented in 3.3.1 to calculate the beam radius at the target.
7. Use an argon-ion laser (488 nm) to excite the internalized Fluo-4 AM dye (λ_EX = 494 nm; λ_EM = 516 nm) and the synchronized LD for the excitation of the endocytosed NRs. Perform the imaging at RT of samples and controls using at least a 40× objective by collecting time-series scans with 256 × 256 pixels/frame resolution in roundtrip mode with a frequency of at least 4 Hz. Perform a recording without any LD excitation to identify any argon-ion laser interference (baseline noise).
8. Remove the background from the data using adaptively weighted penalized least-squares algorithms²¹. Plot the induced Ca²⁺ variations as a function of time. Analyze the transient peaks in the fluorescence intensity using image post-processing software by thresholding at a level three times the standard deviation of the baseline noise (3σ).