Fv1200mpe

Pellets of the pancreatic islets were fixed using formaldehyde (4%, Sigma-Aldrich) overnight. The islets were then washed using PBS. Pellets were centrifuged (1 min at 1300 rpm), the supernatant was removed and agarose (2%, Sigma-Aldrich) was added. Pellets in the agarose were immediately centrifuged (1 min at 1800 rpm). After the agarose solidified, the pellets were transferred into sucrose (30%, Sigma-Aldrich) for overnight incubation at 4 °C. After incubation, the islets were transferred to Tissue-Tek (Sakura, Alphen aan den Rijn, Netherlands) and frozen in methylbutane (Sigma-Aldrich, St. Louis, MO, USA) cooled by liquid nitrogen. Frozen pellets were stored at −80 °C. Sections (20 μm) from the pancreatic islet pellets were cut using a cryomicrotome (Leica CM1950). The samples were stained with diamino-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) and mounted with a vectashield (Vector H-1000, Burlingame, CA, USA) on a glass slide. For confirmation of the nanoparticle signal and its location, the Olympus FV1200MPE (Olympus life Science, Tokyo, Japan) confocal microscope was used (green background - Argon laser λ = 488 nm, DAPI - EPI lamp λ = 405 nm, ICG - LD599 laser λ = 647 nm). The images were taken using 20× (air) and 60× (oil immersion) objectives under 200× or 600× magnification respectively.

Cells were plated in a 24-well plate on cover glasses of 12 mm diameter (Menzel-Gläser, Braunschweig, Germany) 24 h prior to the transfection, which was carried out as described in subsection 2.3. After 24 h transfection, cells were washed with PBS and fixed by incubation with 4% paraformaldehyde (PFA) for 15 min at RT. Next, the cell nuclei were stained with 5 μg/mL of DAPI solution in PBS for 10 min at RT. Afterwards, the cells were washed with PBS and mounted onto glass slides with DAKO fluorescence mounting media (Dako Products, Agilent Technologies, CA, USA). For each condition, three specimens were prepared and at least three images per specimen were obtained. The images were taken using a confocal laser scanning microscope Olympus FV1200MPE (Olympus Corporation, Tokyo, Japan) with ×10 objective and ×60 oil objective at 1024 × 1024-pixel resolution. The images were captured at the wavelengths suitable for detection of DAPI (λex/em = 405/461 nm), EGFP (λex/em = 473/510 nm), and Cy5 (λex/em = 635/664 nm).

We used an inverted laser-scanning microscope (FV1200 MPE, Olympus) optimized for near-infrared (near-IR) throughput and a 25× water objective (XLPlan N, 1.05 N.A., MP, Olympus) with high near-IR transmission for SRS microscopic imaging. A picoEMERALD system (Applied Physics & Electronics) supplied synchronized pulse pump beam (with tunable 720–990 nm wavelength, 5–6 ps pulse width, and 80-MHz repetition rate) and Stokes (with fixed wavelength at 1064 nm, 6 ps pulse width, and 80 MHz repetition rate). Stokes was modulated at 8 MHz by an electronic optic modulator. Transmission of the forward-going pump and Stokes beams after passing through the samples was collected by a high N.A. oil condenser (N.A. = 1.4). A high O.D. bandpass filter (890/220, Chroma) was used to block the Stokes beam completely and to transmit the pump beam only onto a large area Si photodiode for the detection of the stimulated Raman loss signal. The output current from the photodiode was terminated, filtered, and demodulated by a lock-in amplifier at 8 MHz to ensure shot-noise-limited detection sensitivity. The demodulated signal was fed into analog channel of the FV1200 software FluoView 4.1a (Olympus) to form image during laser scanning.