Es1000w camera

HepG2 and SMMC771 cells were labelled with GFP. Chol-let-7a and negative control mimics labelled with Cy5 were purchased from Ribobio (Guangzhou, China).
The GFP-labelled cells (2–3 × 10⁴) were seeded in 8-well BD Falcon™ and BD BioCoat™ Culture Slides (Becton Dickinson Labware, Franklin Lakes, NJ, USA). After 48 h, cells were transfected with Cy5-labelled Chol-let-7a or the negative control mimics (Chol-miRCtrl).
For live-cell imaging, cells were continuously observed using a PerkinElmer UltraVIEW VoX-3D Live Cell Imaging System (Shanghai, China) from 24 to 72 h post-transfection. Digital images were produced using Volocity Demo software (version 5.4, 32-bit). Co-localization events were calculated using the Volocity Demo software as described in the manufacturer’s recommendations. The experiment was repeated 3 times and all samples for each individual experiment were scanned at 5 different locations.
For electron microscopy, cells were collected at 48 h and 60 h after transfection and were fixed with 2.5% glutaraldehyde for 30 min at room temperature, followed by 1.5 h in 2% OsO₄. Samples were stained and examined with a transmission electron microscope (JEOL JEM 1010, Tokyo, Japan), and digital images were obtained with an Erlangshen ES1000W camera (Model 785, Gatan, Warrendale, PA, USA).

Cell death and cellular uptake mechanism were studied by transmission electron microscopy 48 h after irradiation of just after washing (for cell death and uptake mechanism visualization, respectively). A solution of 2% glutaraldehyde + 1% tannic acid in 0.4 M HEPES buffer at pH 7.2 was used for cell fixation (2 h at room temperature). Then, cells were postfixed for 1 h in PBS with 1% osmium tetroxide and 0.8% potassium ferricyanide (Taab Laboratories, England, UK), dehydrated and embedded in Epon. Uranyl acetate and lead citrate were used for double staining of ultrathin sections of samples. Visualization was performed using a JEOL (Tokyo, Japan) JEM-1011 transmission electron microscope complemented with a Gatan Erlangshen ES 1000W camera (Pleasanton, CA, USA).

AFM and TEM analyses were performed at the Microscopy Unit from the University of Valladolid. Samples were deposited on Lacey Carbon Type-A, 300 mesh, copper grids, and visualized and photographed using a JEOL JEM-1011 HR TEM coupled with a Gatan Erlangshen ES1000W camera. For AMF analysis, samples were deposited on a mica surface from aqueous solutions by drop casting. Images were recorded in AC mode (tapping mode) with a CYPHER ES instrument from Asylum Research (Oxford Instruments, Abingdon, UK), using silicon cantilevers AC160TS-R3 with aluminum reflex coating (Olympus) and tip radius <10 nm. The analysis was done using a set point of 500, 72 mV, a drive amplitude of 791.16, a drive frequency of 268.639, and integral gain of 268.639. Data acquisition and control was done with IGOR Pro 6.2 (Asylum Research, Oxford Instruments, Abingdon, UK). Images analysis was done with ARgyle (Argyle Software Ltd., Bath, UK).