Microtime 200 system

For fluorescence correlation spectroscopy (FCS), 0.25 mg of NPs were resuspended in distilled water and incubated with 7 μg of CpG-fluorescein isothiocyanate (FITC) for 60–90 min followed centrifugation and washing at 11,000 × g for 15 min to remove excess CpG ligand. FCS measurements were done using Microtime 200 system from Picoquant GmbH (Berlin, Germany). NPs (approximately 0.25 mg) were diluted in distilled water and 30 μl of solution was dropped onto a 20 × 20 mm No. 1 coverslip (Menzel–Gläser, MA, USA). The focal height was adjusted to 20 μm above this coverslip using an Olympus i × 71 microscope and an Olympus 60 × 1.2 NA objective.

SKOV3 overian carcinoma cell line obtained from American Type Culture Collection (ATCC), Manassas, VA (USA) was grown to 70 % confluence in RPMI supplemented with 10 % FBS and 1 % Pen-Strep. Cells were trypsinized using 0.25 % Trypsin EDTA and seeded on 20 mm round glass-bottom petri dishes. After 24 hours, cells were stained with 500nM of BODIPY for 20 min at 37 °C followed by FLIM imaging on Olympus IX7 microscope. Laser excitation was provided by a pulsed laser diode (PDL-470) emitting 470 nm light and driven by a PDL 828 “Sepia II” driver. This driver was operated at 80 MHz. Measurements were performed on a MicroTime 200 time-resolved, confocal microscope by PicoQuant. The excitation and emission light was focused by a 60× 1.2 NA Olympus objective in an Olympus IX71 microscope, and the emission light was filtered by a 488 long wave pass filter before passing through a 50 µm pinhole. Detection was made by a hybrid photomultiplier assembly. The resolution of the time correlated single photon counting (TCSPC) module was set to 4 ps/bin in order to facilitate the detection at the highest possible resolution. All data analyses were performed using the SymPhoTime software, version 5.3.2. All experimental equipment and the SymPhoTime software were provided by PicoQuant, GmbH as part of the MicroTime 200 system.

Laser excitation was provided by a pulsed laser diode (PDL-470) emitting 470 nm light and driven by a PDL 828 “Sepia II” driver. This driver was operated at 315 KHz. Measurements were performed on a MicroTime 200 time-resolved, confocal microscope by PicoQuant. The excitation and emission light was focused by a 60× 1.2 NA Olympus objective in an Olympus IX71 microscope, and the emission light was filtered by a 488 long wave pass filter before passing through a 50 μm pinhole. Detection was made by a hybrid photomultiplier assembly. The resolution of the time correlated single photon counting (TCSPC) module was set to 512 ps/bin in order to facilitate the detection of the long-lived dye, producing a measurement window around 1.2 μs in length. All data analysis was performed using the SymPhoTime software, version 5.3.2. All experimental equipment and the SymPhoTime software were provided by PicoQuant, GmbH as part of the MicroTime 200 system.