Synthesis of plasmonic nanoparticles
1. Prepare stock solutions with water of the following reagents: silver nitrate (0.005 M), ammonia solution (0.1 M), sodium citrate (1% w/w), sodium borohydride (0.001M), hydrazine monohydrate (0.05 M)
2. At the laboratory temperature (23 °C), add the reagents in the following order and amount into 50 mL beaker while stirring continuously with a magnetic stirrer. Silver nitrate (5 mL), ammonia solution (1.25 mL), sodium citrate (1.25 mL), distilled water (13.425 mL), sodium borohydride (0.075 mL) and hydrazine (4 mL).
3. Stir the solution for 5 minutes and measure UV-VIS absorption spectra
Deposition of silver NP on cell culture plate surfaces
4. Functionalize the cell culture plates by filling up the wells with 1% polyacrylic acid (PAA)
5. After 2 h long functionalization, pour out the PAA from the wells and wash them under running distilled water
6. Coat each well surface by filling up the wells for 2 h with 1% poly(diallyl dimethylammonium chloride) (PDDA)
7. Pour out the unabsorbed PDDA from the wells and wash properly by running distilled water
8. Fill up the wells with AgNPs dispersion and leave still for 45 min to deposit AgNPs on the well surface
9. Empty the wells, wash them with distilled water, and air-dry
Microthermal damage induction in living cells
10. Next day seed the desired cell line (cell line with GFP-tagged protein, such as HSP70, is suitable for direct monitoring of heat shock response of targeted cell) using standard cell culture protocol. Between 80.000 and 100.000 cells per well (depending on cell type) is suitable for optimal confluency for a 24-well plate.
11. Next day put the well plate with cells to the prewarmed (35°C-37°C) confocal microscope and wait about 15 minutes to allow temperature equilibration.
12. Visualize the cells expressing GFP reported using a blue laser (e.g. 488) and appropriate filters.
13. Set carefully the focus of the objective to the plasmon layer by finding the interface between the cell body and the bottom of the well surface. Alternatively, the plasmon layer can be visualized by the transmission light mode.
14. Activate the plasmon layer by 561 nm solid-state laser (or another appropriate laser with a similar wavelength). The amount of emitted heat is regulated by the laser power, pixel dwell time, and the number of irradiation cycles. The appropriate setup must be determined experimentally by the user as there are inevitable differences between various microscopes and lasers.
15. To target heat damage to the defined subcellular region, two approaches can be employed. First, the FRAP-like experiment can be performed. The defined region of interest (ROI) is “bleached” (means exposed to) by 561 nm laser and the cell (with the GFP reporter) is visualized by 488 nm laser. For our FRAP-like experiments where irradiation ROI was pre-defined, the pixel dwell time was fixed at 100 µs, and laser power between 5% to 20% for Alpha Plan-APOCHROMAT 40x water immersion objective. The second approach is based on collinear laser stripes (optimally 16-32 stripes per field). The plasmon layer is activated by 561 nm laser in the pattern of collinear stripes. The pixel dwell time was fixed at 709 µs, and the total irradiation time was 0.85s for one irradiation cycle resulting in 32 colinear stripes across the one microscopic field. See also ref7 for more details for setting up the laser stripping approach in LSM.
16. Visualize cells (GFP-reporter) by 488 nm laser.