Physicochemical characterization of the Turn-ON probes. (A) DLS measurements of the hydrodynamic diameter of P-GFLG-Cy5 (0.5 mg/mL, saline), PC (1 mg/mL, DDW) and PCQ (1 mg/mL, DDW) conjugates. (B) Electron microscopy of (from left to right) a representative TEM image of P-GFLG-Cy5 (1 mg/mL, saline), and representative SEM images of PC (0.01 mg/mL, DDW) and PCQ (0.1 mg/mL, DDW) conjugates. TEM and SEM images are representative of 3 fields of view, with 10 or 20 particles per field, respectively. (C) i. Effect of Cy5 loading on the quenching efficiency of P-GFLG-Cy5 conjugates with different payloads of Cy5. ii. Effect of the addition of quencher molecules to generate FRET PCQ system on the quenching efficacy vs. a self-quenching PC system. All conjugates were measured at the same Cy5-eq. concentration (5 μM). Fluorescence intensity decreased with increasing payload of Cy5 on HPMA copolymer conjugates and when introducing quencher molecules to generate PCQ conjugate. Filter set: λEx: 630 nm, filter: 665 nm, λEm: 670 nm. The data are presented as mean ± SD (n = 3).