Figure 1.
Overview of the radioluminescence microscope.
(A) Radioluminescence is produced within a scintillator plate following the emission of a beta particle from a radiotracer within a cell (yellow glow). The optical photons are captured by a high-numerical-aperture objective coupled to a deep-cooled EM-CCD camera. Emission and excitation filters used in combination with a light source allow for concurrent fluorescence and brightfield microscopy. (B) Photograph of the system showing a glass-bottom dish containing a scintillator plate immersed in cell culture medium and placed into the inverted microscope. (C) Three GFP-expressing HeLa cells located near the corner of a scintillator plate were localized using fluorescence microscopy (arrows). The edge of the scintillator plate is outlined in red. (D) After incubation with FDG (400 µCi, 1 h), these three cells also produced focal radioluminescence signal coincident with the fluorescent emission.
Figure 2.
Radioluminescence imaging of FDG uptake in single cells.
Human breast cancer cells (MDA-MB-231) were deprived of glucose for 1 h, incubated for 1 h with FDG (400 µCi) and 2-NBDG (100 µM), and then washed. (A) Brightfield (scale bar, 100 µm.), radioluminescence (FDG), and fluorescence (2-NBDG) micrographs (Objective: 40X/1.3 NA). Overlay, showing co-localized radioluminescence (green) and fluorescence (red). (B) Scatter plot comparing FDG and 2-NBDG uptake, computed over 140 cells (light red dots) and 26 control ROIs (blue dots). The green line was obtained by linear regression (correlation, r = 0.74). Arbitrary units (A.U.). (C) Radioluminescence (FDG) and fluorescence (2-NBDG) intensity shown along a line profile [red dashed line in (A)].
Figure 3.
Dynamic radioluminescence imaging of FDG in single cells.
Micrographs (brightfield and radioluminescence) were acquired every 6 min for 8 h for three experiments. (A) MDA-MB-231 cells are imaged while being incubated with FDG (5 µCi). (B) Glucose (25 mM) is added 2 h after the beginning of the incubation with FDG (5 µCi). (C) FDG is withdrawn at the start of imaging after incubation (1 h, 400 µCi). Scale bar: 100 µm. (E–F) Time-activity curves plotted for individual cells (light red lines) and 10 control ROIs manually selected in the background (light blue lines), for all three experiments. The thick red and blue lines represent the average for cells and control ROIs, respectively.
Figure 4.
Pharmacokinetics analysis in single cells.
(A) Two-tissue compartmental model describing FDG pharmacokinetics, including influx (K1), efflux (k2), phosphorylation to FDG-6-phosphate (k3), and dephosphorylation (k4). (B) Patlak analysis modeling FDG influx kinetics for a single cell (highlighted by a red circle in Figure 3A). (C,D) Rate of efflux (k2) and phosphorylation (k3) plotted as a function of rate of influx (K1) for all the cells in the microscope’s field of view. (E) Compartmental analysis modeling FDG efflux kinetics from a single cell (highlighted by a red circle in Figure 3C) after withdrawal of FDG, presenting a fast and a slow component. (F) The model for FDG efflux is the sum of a fast and a slow component (rates λ1 and λ2, respectively), which are plotted for all the cells in the field of view.
Figure 5.
Radioluminescence imaging of gene expression in single cells.
Human cervical cancer cells (HeLa) transfected with a fusion PET/fluorescence reporter gene were incubated with FHBG (300 µCi, 2 h). (A) Brightfield (scale bar, 50 µm), radioluminescence (FHBG), and fluorescence (RFP) micrographs (objective, 100X/1.35 NA). Overlay shows FHBG radioluminescence (green), RFP fluorescence (red), and cell outline segmented from brightfield. Cells negative for RFP are also negative for FHBG (red arrows). (B) Same as (A), but with a 40X/1.3 NA objective (scale bar, 100 µm). White arrows indicate cells with weak fluorescence intensity but substantial radioluminescence intensity. The green arrow points to a cell with no RFP expression but ambiguous radioluminescence intensity. (C) Scatter plot of FHBG vs. RFP uptake, computed for 245 cells (light red dots) and 100 control ROIs (blue dots). Arbitrary units. (D) Radioluminescence and fluorescence shown along a line profile [red dashed line in (A)]. (E) Same experiment as (A,B), but using control wild-type HeLa cells (scale bar, 100 µm).
Figure 6.
FDG aggregates were obtained by evaporating an aqueous solution of FDG between a scintillator and a glass-bottom imaging dish. (A) Fused radioluminescence and brightfield images; (B) Brightfield and (C) radioluminescence images, magnified; (D) Brightfield and (E) radioluminescence images, further magnified, focusing on one particular FDG aggregate; (F,G) 2-D Gaussian fit of (D) and (E), respectively. (H) Radioluminescence microscope sensitivity, obtained by imaging the decay of a drop of FDG (2.6 µCi) over time. Solid line: mean pixel intensity; Dashed line: ideal exponential decay for 18F. (I) Per-pixel signal-to-noise ratio, defined as the ratio of the average pixel intensity to the noise standard deviation. The sensitivity of the system is defined here as the amount of activity required per area to achieve a SNR of 5 (Rose criterion ).