Positron emission particle tracking (PEPT) offers high spatiotemporal resolution for 3D localization and tracking of a single moving particle. Unlike PET, which images the average distribution of many radiotracers, PEPT uses the γ-rays from a single radiolabeled particle to pinpoint its location. Currently, PEPT is primarily used in industrial settings to study particle-fluid or particle-particle interactions. However, its high spatiotemporal resolution makes it highly promising for biomedical applications, particularly in studying blood flow dynamics in cardiovascular disease and cancer. In vivo PEPT could provide breakthroughs in understanding complex multiphase blood flow, crucial for clinical physiology and drug delivery. Furthermore, tracking single radiolabeled cells could offer detailed insights into cell migration and interactions with vessels and tissues, information unavailable from standard clinical imaging techniques which only provide average cell location. The major limitation preventing biomedical in vivo PEPT has been the lack of methods to radiolabel and isolate single particles of suitable composition, size, and radioactivity concentration (specific activity). While one study tracked a single cancer cell using whole-body PET, the low specific activity limited its application to high-speed flows. Inorganic particles offer advantages over cells as tracers due to higher resistance to radiolysis and controllable physicochemical properties. However, challenges remain in finding suitable material-radionuclide combinations, developing radiolabeling methods to achieve high specific activity, and establishing methods to isolate and manipulate single sub-micrometre particles.

Existing literature highlights the potential of PEPT for various applications. Studies demonstrate its use in industrial settings such as evaluating particle-fluid or particle-particle interactions in turbulent and dense flows. The potential for biomedical applications, particularly the study of blood flow dynamics, is highlighted by the capabilities of systems like the Forte camera, capable of tracking particles moving at 1 m s⁻¹ to within 0.5 mm approximately 250 times a second. Recent advancements like the superPEPT camera are expected to further improve spatiotemporal resolutions. A crucial study demonstrated the feasibility of PEPT in biomedicine by tracking a single radiolabeled cancer cell using whole-body PET, showcasing the potential of the technique. However, this study also revealed limitations inherent in using living cells as tracers, particularly the restriction on radioactivity levels to prevent cell damage. This necessitates the exploration of alternative tracers like inorganic particles, offering the potential for higher radioactivity concentrations without compromising viability.

This study aimed to develop the first single radiolabeled particle for biomedical PEPT. Various material-radionuclide combinations were evaluated using chelator-based and non-chelator radiolabeling strategies. ⁶⁸Ga-silica was identified as the optimal combination due to ⁶⁸Ga's high affinity for silica. The researchers synthesized highly homogeneous sub-micrometre silica particles (smSiP) using a modified Stöber method, confirmed by scanning electron microscopy (SEM), ζ-potential measurements, energy-dispersive X-ray spectroscopy (EDS), and Fourier-transform infrared spectroscopy (FT-IR). The radiolabeling method was optimized to maximize specific activity. A cation exchange method was used to concentrate the ⁶⁸Ga eluate, improving radiolabeling yield (RLY) to 90–100% for typical particle concentrations. Flow cytometry was employed to quantify particle concentration and control the number of particles precisely. A three-step purification protocol using decreasing concentrations of EDTA removed unreacted and colloidal ⁶⁸Ga, achieving high radiochemical purity (RCP). A fractionation protocol was developed to isolate single radiolabeled particles. The specific activity of the isolated particles was determined. In vitro PET/CT imaging was performed to assess the scanner's sensitivity. The behavior of single particles during injection was studied to evaluate potential particle loss. In vivo PET/CT and real-time PEPT imaging were conducted in healthy BALB/c mice. To address the potential impact of particle surface properties, the study was extended to include PEGylated silica particles, comparing their behavior with uncoated particles. Ex vivo biodistribution studies were conducted to assess organ uptake and autoradiography was used for precise localization of the single particle within the lung tissue. The Birmingham method was used for PEPT analysis of the list-mode PET data, allowing for the quantification of particle velocity and movement.

The study successfully synthesized highly homogeneous, non-porous silica particles (950 ± 50 nm) with excellent radiolabeling properties using ⁶⁸Ga. A novel method for concentrating the ⁶⁸Ga eluate from a generator significantly enhanced the radiolabeling efficiency. The use of flow cytometry enabled precise quantification of particle concentration, crucial for controlling the number of particles in radiolabeling reactions and successfully isolating single particles with a specific activity of 2.1 ± 1.4 kBq per particle—approximately 100 times higher than previously reported for single cells. In vivo PET/CT imaging demonstrated clear visualization of the single particle in the lungs within 5 minutes post-injection using kBq levels of radioactivity (three orders of magnitude lower than conventional PET). Real-time PEPT tracking successfully quantified particle velocity and movement, providing insights into blood flow and breathing motion. The rapid lung uptake of the uncoated particles was attributed to the formation of a protein corona, increasing size and restricting capillary flow. PEGylation increased the specific activity to 3.1 ± 3.2 kBq per particle, but did not prevent rapid lung uptake, likely due to the particle size or single-particle nature which increases susceptibility to phagocytosis. Ex vivo biodistribution studies and autoradiography confirmed the presence of a single radioactive hotspot in the lung, and quantitative data across different stages (injection, biodistribution, lung slice analysis, autoradiography) were highly consistent.

This study addresses the critical challenge of developing suitable single-particle tracers for in vivo PEPT, paving the way for its application in biomedical research. The high specific activity achieved, coupled with the successful in vivo imaging and real-time tracking, demonstrates the potential of this approach for studying hemodynamics in various settings. The choice of silica as a particle material proves advantageous due to its biocompatibility, stability, and radiolabeling properties. The use of flow cytometry for precise particle quantification revolutionizes the process of isolating single radiolabeled particles. The rapid lung uptake observed with both coated and uncoated particles necessitates further investigation into particle size, flexibility, and strategies to modulate the phagocytic response. While the current study focuses on the lung, future studies can expand to other organs and physiological systems. The observed bias in particle position due to tissue density variations requires further evaluation to optimize PEPT algorithms. The use of low radioactivity doses (0.4-2.9 kBq) showcases the technique’s potential for minimal invasiveness and reduced toxicity.

This work successfully demonstrated in vivo real-time PEPT imaging using a single radiolabeled sub-micrometre silica particle. The high specific activity achieved (2.1 ± 1.4 kBq per particle) enabled clear visualization and tracking with significantly lower radioactivity doses than conventional PET. The results are highly promising for future applications in evaluating blood flow dynamics and assessing organ motion, particularly in disease settings like cardiovascular conditions and cancer. Further research is needed to optimize particle design and address limitations related to lung uptake and PEPT algorithm accuracy for improved spatial resolution. The combination of PEPT with total-body PET scanners holds significant promise for the future of quantitative whole-body hemodynamic studies.

The rapid lung uptake observed in both coated and uncoated particles remains a limitation, necessitating further investigation into optimizing particle surface properties to extend circulation time. The current study focused on healthy mice; further studies in disease models are needed to assess the technique’s clinical translation potential. The positioning error in PEPT analysis, particularly at early timepoints, should be considered when interpreting the velocity data. The limited number of animals in the PEGylated particle studies warrants further investigation with a larger cohort. The potential bias introduced by tissue heterogeneity on the accuracy of particle positioning in PEPT needs further examination and algorithm refinement.