300 mesh carbon formvar coated copper grid

Purified complexes were diluted to 0.03 mg/ml in 20 mM Tris, 300 mM NaCl, pH 8 buffer, and 2.5 µl were loaded for 30 s onto a 300 mesh carbon/formvar coated copper grid (Agar Scientific). After Blotting the excess, the grid was negatively stained with 1% aqueous uranyl acetate for 45 s and analyzed using a Philips CM200-FEG transmission electron microscope operating at 200 kV equipped with a TVIPS TemCam-F224HD CCD camera and TVIPS EM-Menu4, EM tools, and EM-SPC software packages. The micrographs were acquired at a magnification of 50,000×, corresponding to a pixel size of 3.3 Å/pixel on the specimen. As the complexes assumed a preferred orientation onto the grid, dataset of around 2000 untilted images and a set of 25 RCT pairs of images were collected at −55° and 0° for each sample.

Er-UCNPs (NaYF₄: 17% Yb³⁺, 3% Er³⁺) and Tm-UCNPs (NaYF₄: 20% Yb³⁺, 0.5% Tm³⁺) were synthesized in-house with high-temperature co-precipitation in organic oils as described earlier [32 (link)]. The oleic acid–capped UCNPs were characterized by transmission electron microscopy (TEM), by dispersing to 0.3 mg/ml in toluene and applying 3 µl on 300-mesh carbon-formvar-coated copper grid (Agar Scientific, Essex, UK), and imaging with JEM-1400 Plus (JEOL, MA, USA) under 80-kV electron beam. UCNP size was measured from TEM images with semi-automatic in-house program based on setting color shade thresholds. Emission spectra of UCNPs were measured with a Varian Cary® spectrophotometer modified with a 980-nm excitation laser source from 1 mg/ml dilutions of oleic acid–capped UCNPs in DMSO. The UCNPs were polyacrylic acid (PAA, Mw 2000) coated and bioconjugated following a previously published protocol with a small modification [33 ]. Er-UCNPs were conjugated with anti-CA125 Mab 4602 and Tm-UCNPs were conjugated with anti CA15-3 Mab Ma552 using EDC/sulfo-NHS chemistry. For anti-CA125, the EDC/sulfo-NHS activation was done with 2.5 mM EDC and for anti-CA15-3 the activation was done with 12.5 mM EDC.