Single-cell imaging

Adherent MDA-MB-231 cells (5 × 10⁴) were seeded sparsely on a tissue-culture-treated 35 mm glass-bottom dish (ibidi Labware GmbH, Germany). The following day, MDA-MB-231 cells were incubated with [¹⁸F]HFB or [¹⁸F]FDG (250 µCi/mL) for 30 min at 37^°C, whilst suspension MDA-MB-231 cells were obtained by tryptinizing prior to radiolabelling. In addition to [¹⁸F]HFB or [¹⁸F]FDG, cells were co-incubated with live/dead nuclei fluorescent stain for mammalian cells (NucBlue Live ReadyProbe and NucGreen Dead 488 ReadyProbe; Thermo Fisher Scientific) and/or DiIC₁₈(5) solid (1,1′-Dioctadecyl-3,3,3′,3′ Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt) membrane dye (Thermo Fisher Scientific). The cells were washed twice with PBS and suspension MDA-MB-231 as well as Jurkat T cells were re-suspended in PBS (1 mL) and aliquoted in a tissue-culture-treated 35 mm glass-bottom dish. Fluorescence images were initially acquired to confirm cell viability and identify a region containing both live cells, clusters of dead cell nuclei and membrane fragments. The DAPI channel was used for cell nuclei (excitation/emission: ‎358 nm⁄461 nm), the GFP channel for dead cell nuclei (469 nm/525 nm) and the Texas Red channel for cellular membrane (559 nm/630 nm).

Single-cell resolution radioluminescence imaging was performed on cells labelled with [¹⁸F]HFB or [¹⁸F]FDG using a previously described protocol [31]. Briefly, a transparent scintillating crystal (1 cm × 1 cm × 0.5 mm CdWO₄, both sides polished; MTI Corp., Richmond, CA) was submerged in an imaging dish and gently positioned on top of the radiolabelled cells. Scintillation was generated by emission of positrons from the radiolabelled cells and captured using a low-light microscope with an effective magnification of 5×. The low-light microscope was comprised of a 20× magnification and 0.75 NA microscope objective lens (CFI Plan Apochromat λ; Nikon Instruments Inc., Melville, NY), a 4× magnification, 0.2 NA tube lens (CFI Plan Apochromat λ; Nikon Instruments Inc., Melville, NY) and deep-cooled electron-multiplying charge-coupled device (EMCCD) camera (ImageEM C9100-13, Hamamatsu Photonics K.K., Japan). Fluorescence and brightfield images were captured with 5× effective magnification and no pixel binning (3.2 × 3.2 µm/px resolution). The microscope was first focused on the cell nucleus using the DAPI channel. The focus was then shifted ~5 µm towards the scintillator edge. The radioluminescence images were acquired using an EM gain of 1200×, 4×4 pixel binning (12.8 × 12.8 µm/px resolution), an exposure time of 9 - 160 ms and 10,000 image frames. To compare different radioluminescence images, the radioactive decay counts were normalised by the total acquired exposure time, i.e., the number of frames multiplied by the exposure time per frame.