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Single-cell radioluminescence microscopy with two-fold higher sensitivity using dual scintillator configuration.

ABSTRACT: Radioluminescence microscopy (RLM) is an imaging technique that allows quantitative analysis of clinical radiolabeled drugs and probes in single cells. However, the modality suffers from slow data acquisition (15-30 minutes), thus critically affecting experiments with short-lived radioactive drugs. To overcome this issue, we suggest an approach that significantly accelerates data collection. Instead of using a single scintillator to image the decay of radioactive molecules, we sandwiched the radiolabeled cells between two scintillators. As proof of concept, we imaged cells labeled with [18F]FDG, a radioactive glucose popularly used in oncology to image tumors. Results show that the double scintillator configuration increases the microscope sensitivity by two-fold, thus reducing the image acquisition time by half to achieve the same result as the single scintillator approach. The experimental results were also compared with Geant4 Monte Carlo simulation to confirm the two-fold increase in sensitivity with only minor degradation in spatial resolution. Overall, these findings suggest that the double scintillator configuration can be used to perform time-sensitive studies such as cell pharmacokinetics or cell uptake of short-lived radiotracers.

SUBMITTER: Kim TJ

PROVIDER: S-EPMC7340323 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Single-cell radioluminescence microscopy with two-fold higher sensitivity using dual scintillator configuration.

Kim Tae Jin TJ Wang Qian Q Shelor Mark M Pratx Guillem G

PloS one 20200707 7

Radioluminescence microscopy (RLM) is an imaging technique that allows quantitative analysis of clinical radiolabeled drugs and probes in single cells. However, the modality suffers from slow data acquisition (15-30 minutes), thus critically affecting experiments with short-lived radioactive drugs. To overcome this issue, we suggest an approach that significantly accelerates data collection. Instead of using a single scintillator to image the decay of radioactive molecules, we sandwiched the rad ...[more]

PMID: 32634153

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Project description:UnlabelledThe radiotracer 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) is commonly used to measure cell proliferation in vivo. As a marker of cell proliferation, (18)F-FLT is expected to be differentially taken up by arrested and actively dividing cells, but PET measures only aggregate uptake by tumor cells and therefore the single-cell distribution of (18)F-FLT is unknown. We used a novel in vitro radioluminescence microscopy technique to measure the differential distribution of (18)F-FLT radiotracer with single-cell precision.MethodsUsing radioluminescence microscopy, we imaged the absolute uptake of (18)F-FLT in live MDA-MB-231 cells grown under different serum conditions. We then compared (18)F-FLT uptake with a standard measure of cell proliferation, using fluorescence microscopy of 5-ethynyl-2'-deoxyuridine incorporation in fixed cells.ResultsAccording to 5-ethynyl-2'-deoxyuridine staining, few cells (1%) actively cycled under serum deprivation whereas most of them (71%) did under 20% serum. The distribution of (18)F-FLT reflected this dynamic. At 0% serum, uptake of (18)F-FLT was heterogeneous but relatively low. At 20% serum, a subpopulation of (18)F-FLT-avid cells, representing 61% of the total population, emerged. Uptake of (18)F-FLT in this population was 5-fold higher than in the remainder of the cells. Such a dichotomous distribution is not typically observed with other radiotracers, such as (18)F-FDG.ConclusionThese results suggest that increased (18)F-FLT uptake by proliferating cells is due to a greater fraction of (18)F-FLT-avid cells rather than a change in (18)F-FLT uptake by individual cells. This finding is consistent with the fact that (18)F-FLT uptake is mediated by thymidine kinase 1 expression, which is higher in actively dividing cells. Overall, these findings suggest that, within the same patient, changes in (18)F-FLT uptake reflect changes in the number of actively dividing cells, provided other parameters remain the same.

Project description:PURPOSE:Cell-based therapies are showing great promise for a variety of diseases, but remain hindered by the limited information available regarding the biological fate, migration routes and differentiation patterns of infused cells in trials. Previous studies have demonstrated the feasibility of using positron emission tomography (PET) to track single cells utilising an approach known as positron emission particle tracking (PEPT). The radiolabel hexadecyl-4-[18F]fluorobenzoate ([18F]HFB) was identified as a promising candidate for PEPT, due to its efficient and long-lasting labelling capabilities. The purpose of this work was to characterise the labelling efficiency of [18F]HFB in vitro at the single-cell level prior to in vivo studies. PROCEDURES:The binding efficiency of [18F]HFB to MDA-MB-231 and Jurkat cells was verified in vitro using bulk gamma counting. The measurements were subsequently repeated in single cells using a new method known as radioluminescence microscopy (RLM) and binding of the radiolabel to the single cells was correlated with various fluorescent dyes. RESULTS:Similar to previous reports, bulk cell labelling was significantly higher with [18F]HFB (18.75 ± 2.47 dpm/cell, n = 6) than 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) (7.59 ± 0.73 dpm/cell, n = 7; p ? 0.01). However, single-cell imaging using RLM revealed that [18F]HFB accumulation in live cells (8.35 ± 1.48 cpm/cell, n = 9) was not significantly higher than background levels (4.83 ± 0.52 cpm/cell, n = 12; p > 0.05) and was 1.7-fold lower than [18F]FDG uptake in the same cell line (14.09 ± 1.90 cpm/cell, n = 13; p < 0.01). Instead, [18F]HFB was found to bind significantly to fragmented membranes associated with dead cell nuclei, suggesting an alternative binding target for [18F]HFB. CONCLUSION:This study demonstrates that bulk analysis alone does not always accurately portray the labelling efficiency, therefore highlighting the need for more routine screening of radiolabels using RLM to identify heterogeneity at the single-cell level.

Dataset Information

Single-cell radioluminescence microscopy with two-fold higher sensitivity using dual scintillator configuration.

Publications

Single-cell radioluminescence microscopy with two-fold higher sensitivity using dual scintillator configuration.

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