3.1 Pulsed laser annealing of gold films
The laser system is a Q-switched, frequency-doubled Nd:YAG laser at 532 nm in wavelength and 6 ns in pulsewidth (Continuum, Minilite I). The laser beam is linearly polarized and the pulse energy can be adjusted using a variable attenuator consisting of a half wave plate and a polarizing beam splitter. The pulse energy was measured using a pyroelectric optical energy detector (Newport, 1918-C). The sample was prepared by depositing a 10 nm thick gold film and a 1 nm titanium adhesion layer on a glass coverslip via electron beam evaporation. During pulsed laser annealing, the laser beam was sent through a holographic diffuser (Edmund Optics, NT55-848) and irradiated the gold film at a normal incidence. The diffuser reduces interference effects and smoothens the laser intensity profile. The laser spot, with a 3 mm diameter, scanned over the entire area of the gold film at a moving speed of 0.5 mm/sec. Using a laser pulse repetition rate of 10 Hz, the typical number of pulses received per unit area was 60. After annealing, the sample morphology was inspected with a scanning electron microscope (Hitachi S4700 field emission SEM). The gold particle diameter and density were calculated using image analysis software (ImageJ, http://rsbweb.nih.gov/ij/index.html
3.2 Patterned laser irradiation and a time-resolved imaging system
illustrates the experimental setup. The laser source is the same one used for gold film annealing as described in 3.1. A shadow mask with a rectangular opening (2.3×4 mm) was placed in the laser beam path to cast the desired optical pattern. The laser pattern was imaged through an objective lens (40×, 0.6 NA) onto the device at a 25× reduction. Cell cultures were observed using an inverted microscope (Zeiss, AxioObserver) with phase contrast and epi-fluorescence capabilities. To capture the extremely short-lived cavitation bubbles induced by the pulsed laser (typically with a lifetime < 1 μs), a time-resolved imaging system was constructed. This included a high-speed Intensified CCD camera (Princeton Instrument, PI-MAXII), providing exposure times as short as 500 ps. A programmable delay between receiving the laser triggering signal and the camera shutter opening was set by the camera control unit. After the polarizing beam splitter, one arm of the laser beam was sent through a fluorescent dye cell (Exciton, LDS 698). The excited fluorescence pulse (wavelength centered around 698 nm) was coupled into a multimode fiber (Thorlabs, BFL37-600) and then sent through the microscope condenser to illuminate the sample, in synchronization with the camera shutter. A nanosecond time delay between the captured bubble image and the excitation laser pulse can be achieved by controlling the length of the optical fiber delay line.
Schematic of the experiment setup for light-patterned molecular delivery and the time-resolved imaging system used to capture the cavitation bubble dynamics.
3.3 Dye delivery efficiency and cell viability evaluations
HeLa and immortalized human embryonic kidney cells (HEK293T) were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (Hyclone), 50 mg/ml non-essential amino acids, 50 mg/ml penicllin/streptomycin, and 50 mg/ml sodium pyruvate. Cells were harvested and plated in a dish on top of a gold particle coated coverslip, fabricated by pulsed laser annealing. Cells were incubated overnight until about 90-100% cell confluence was reached. Cell density was measured and controlled to be around 3700 cells/mm2 for HeLa and 4000 cells/mm2 for HEK 293T. During laser pulsing, the cells were immersed in cell culture medium containing the membrane-impermeable fluorescent dye calcein (Invitrogen, 0.62 kDa) at 100 µM. After cavitation induction, the cell culture was washed with phosphate buffered saline (PBS, pH 7.4) and incubated in fresh medium for 90 minutes at 37°C before adding propidium iodide (PI) at 5 µg/ml. PI is used as a dead cell indicator as it stains the DNA of dead cells and is excluded from viable cells. Cells were incubated in the presence of PI for another 30 minutes at room temperature and were washed with PBS, pH 7.4, before fluorescence imaging. Dye delivery efficiency was measured as the percentage of cells showing obvious calcein uptake within the laser irradiation pattern. Viability was calculated as the percentage of cells that were negative of PI staining within the laser irradiation pattern.
For multiplexed molecular delivery, the cell culture was first irradiated with the horizontal bar pattern to induce dextran-tetramethylrhodamine (Invitrogen, 40 kDa) uptake. Following a wash and change of medium, the same culture was irradiated with the two vertical bars to induce calcein uptake. The cell culture was then washed and checked for fluorescence. A concentration of 100 µM was used for both fluorescent molecules.