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# Performing radiosynthesis in microvolumes to maximize molar activity of tracers for positron emission tomography

## Abstract

Positron emission tomography (PET) is a molecular diagnostic imaging technology to quantitatively visualize biological processes in vivo. For many applications, including imaging of low-tissue density targets (e.g., neuroreceptors), imaging in small animals, and evaluation of novel tracers, the injected PET tracer must be produced with high molar activity to ensure low occupancy of biological targets and avoid pharmacologic effects. Additionally, high molar activity is essential for tracers with lengthy syntheses or tracers transported to distant imaging sites. Here we show that radiosynthesis of PET tracers in microliter volumes instead of conventional milliliter volumes results in substantially increased molar activity, and we identify the most relevant variables affecting this parameter. Furthermore, using the PET tracer [18F]fallypride, we illustrate that molar activity can have a significant impact on biodistribution. With full automation, microdroplet platforms could provide a means for radiochemists to routinely, conveniently, and safely produce PET tracers with high molar activity.

## Introduction

Positron emission tomography (PET) is an effective in vivo molecular imaging technology in clinical care, preclinical and clinical research, and drug discovery1,2,3,4,5. PET applications span a broad range of medical fields, including oncology, cardiology, immunology, and neurology. In the latter, for example, PET is used in the diagnosis and monitoring of neurodegenerative disorders6, and is a vital tool in the discovery and development of novel therapeutics by enabling in situ measurements of drug target occupancy, binding kinetics, etc.7. Based on the principles of “tracer kinetics”, PET enables measurements of biological processes such as receptor occupancy, cell proliferation, metabolism, apoptosis, and gene expression, quantification of their temporal changes, and identification of their roles in the mechanisms of disease without perturbing the underlying biology8, 9. While PET tracers have been developed incorporating various radionuclides10, those labeled with fluorine-18 are of particular interest due to the wide availability of this radionuclide and its favorable physical and chemical properties11.

During synthesis of PET tracers, inevitable contamination by sources of non-radioactive [19F]fluoride leads to a final product comprising a mixture of 18F- and 19F-labeled molecules, of which only the former are detectable by the PET scanner. Molar activity (Am; commonly referred to as “specific activity”) describes the ratio of the radioactivity (i.e., quantity of 18F-labeled molecules) to the total quantity of radioactive plus non-radioactive (i.e., 19F-labeled) molecules12,13,14. High Am of PET tracers is critical for many applications15,16,17: it minimizes occupancy when imaging low tissue density targets (e.g., neuroreceptors)5, 18,19,20,21, facilitates PET “microdosing” studies to evaluate the pharmacokinetics of novel drug compounds22,23,24,25, extends the distance/time over which tracers can be transported to the imaging site26, and is critical in preclinical research, where much higher concentrations of the tracer per mass of the animal must be injected (compared to humans) to achieve sufficient signal-to-noise ratio19, 20, 27.

The theoretical maximum Am of a radiofluorinated tracer is $$A_{\rm m}^{{\rm max}}$$ = 1710 Ci/µmol [63.3 TBq/µmol] (i.e., all molecules are labeled with 18F). However, reported Am values are most typically in the 0.5–1.0 Ci/µmol [19–37 GBq/µmol] range (for nucleophilic radiofluorination)26, though there is considerable variation28 and a few reports of significantly higher Am. Though there is not a consensus on what constitutes “high” Am, we consider values >5 Ci/µmol [>185 GBq/µmol] to be “high”. The discrepancy of three orders of magnitude between typical and maximum theoretical Am can be attributed to [19F]fluoride contamination in the radioisotope solution (i.e., from cyclotron production and delivery process) and/or from the radiosynthesis process itself (i.e., from reagents and/or synthesis apparatus). Since [18F]fluoride and [19F]fluoride have the same reactivity, both are incorporated into the precursor nearly in proportion to their abundance. Thus, the molar activity of a tracer can be expressed as:

$$A_{\rm{m}} = A_{\rm{m}}^{\rm{max}}\left( {\frac{{\,^{18}{\rm{F}}_{\rm{c}}}}{{\,^{18}{\rm{F}}_{\rm{c}} + \,{}^{19}{\rm{F}}_{\rm{c}} + \,{}^{19}{\rm{F}}_{\rm{s}}}}} \right)$$
(1)

where 18Fc is the number of [18F]fluoride ions from the cyclotron, 19Fc is the number of [19F]fluoride ions from the cyclotron, and 19Fs is the number of [19F]fluoride ions from the synthesis process.

The typical strategy to increase Am is to start with a higher amount of radioactivity (e.g., >1 Ci [>37 GBq]), thus increasing the numerator in Eq. 1. In this approach, Am > 20 Ci/µmol [>740 GBq/µmol] at end of synthesis (EOS) has been reported from 5 Ci [185 GBq]29, 30, and unusually high Am values of 130–160 Ci/µmol [4.7–5.9 TBq/µmol] at EOS were recently reported starting with ~6 Ci [220 GBq]31. However, the use of such large amounts of radioactivity poses safety challenges in production and handling of the tracer, and can lead to significant radiolysis of the product32. Furthermore, the costs associated with using higher amounts of radioactivity may be difficult to justify if the goal is only to perform a small number of patient scans or to perform preclinical imaging. As an aside, another approach for increasing molar activity that is not reflected in Eq. 1 is the addition of multiple labeling sites per tracer molecule16. Using trifluoroborate derivatives, tripling of molar activity has been reported33.

Reducing the amount of 19F-contamination (i.e., 19Fc and 19Fs in Eq. 1) is another strategy to improve Am. Contamination present in the [18F]fluoride solution after cyclotron bombardment can be characterized by:

$$A_{\rm m}^{{\rm isotope}} = A_{\rm m}^{{\rm max}}\left( {\frac{{\,^{18}\rm{F}_{\rm c}}}{{\,^{18}\rm{F}_{\rm c} + \,{}^{19}F_{\rm c}}}} \right)$$
(2)

Very high values of $$A_{\rm m}^{{\rm isotope}}$$ have been reported after optimization of cyclotron bombardment conditions (140 Ci/µmol [5 TBq/µmol] at EOB)34 or processes related to loading and cleaning the [18O]water target (1200 Ci/µmol [44 TBq/µmol] at EOB)35. However, it has been remarked that typical values are generally closer to the range of 10 Ci/µmol [370 GBq/µmol] at EOB15 or 15–30 Ci/µmol [560–1100 GBq/µmol] at EOB17, 36. Though 19FC can likely be reduced (and $$A_{\rm m}^{{\rm isotope}}$$increased) for most cyclotrons, most radiochemists will not have control over this aspect of the tracer production. Contamination from the synthesis process (19Fs) is generally even more significant and thus dominates the molar activity of the final tracer. In one study, radiolysis of Teflon was found to be a major source, with 90–95% reduction of contamination by avoiding Teflon (tubing, stir bars, etc.)37. Other sources of contamination include reagents (such as K2CO3, solvents, etc.)38, and anion exchange cartridges (e.g., quaternary methylammonium, QMA) that are often used in the first step of 18F-radiosyntheses39.

We hypothesize that 19F-contamination could be significantly decreased (and Am increased) by replacing conventional radiochemistry approaches with microfluidic chips, due to orders of magnitude reduction in volumes and surface areas. Recently, droplet-based radiochemistry systems operating at the microliter scale have been developed40, 41. Despite the small volumes, these chips can produce clinically relevant amounts of PET tracers by pre-concentrating the radioisotope42. A few preliminary results have suggested that Am of tracers produced with this platform may be significantly higher than macroscale methods40, 43,44,45,46. Here, we report on a comprehensive study of these differences in Am, comparing radiosyntheses of two model compounds performed at the micro- and macroscales. We observed interesting trends that shed light on the relative magnitude of the various contributions of 19F-contamination. Microscale radiochemistry, it turns out, provides intrinsic advantages compared to conventional methods and could provide a means for consistently achieving high Am without requiring the high starting activities that are necessary in macroscale processes.

## Results

### Factors affecting molar activity

Micro- and macroscale radiosynthesis were compared using two model precursors: 1,4-dinitrobenzene for aromatic fluorination leading to 1-[18F]fluoro-4-nitrobenzene ([18F]FNB), and tosyl-fallypride for aliphatic fluorination leading to [18F]fallypride, a tracer for dopamine D2/D3 receptors (Fig. 1). The microscale synthesis was performed on a microdroplet synthesis platform (Fig. 2), and the macroscale synthesis, with a commercial synthesizer (ELIXYS, Sofie Biosciences, Inc.). The experimental design is illustrated in Fig. 3. To facilitate comparisons among different experiments, molar activity values in the following sections have been corrected to end of bombardment (EOB).

### Effect of reaction volume

Suspecting that increasing amount of reagents (i.e., precursor, base, phase transfer catalyst) would increase [19F]fluoride contamination, we performed experiments in which the amount of [18F]fluoride/[18O]H2O was held constant, but the amount of all reagents was varied (proportionally). The amount of base/phase transfer catalyst was varied by adding different volumes of the stock solutions during the [18F]fluoride drying process, and the amount of precursor was varied in proportion by adding different volumes of the stock solution. Indeed, macroscale syntheses performed with increasing reaction volumes from 0.1 to 3.0 mL showed decreasing molar activity (Fig. 4a). For example, for [18F]FNB, Am resulting from 0.1 mL reaction volume was nearly 10× higher than for the 3.0 mL reaction, i.e., 0.41 ± 0.04 Ci/µmol [15.2 ± 1.5 GBq/µmol] (n = 3) vs. 0.04 ± 0.03 Ci/µmol [1.5 ± 1.1 GBq/µmol] (n = 3), respectively. The same synthesis (starting with similar amount of radioactivity) in the microchip produced much higher Am, 22.7 ± 0.6 Ci/µmol [840 ± 22 GBq/µmol] (n = 12), and Am was nearly constant despite fluorination volume variation from 2 to 8 µL (Fig. 4b). For [18F]fallypride, Am was also found to be nearly constant (18.2 ± 0.4 Ci/µmol [673 ± 15 GBq/µmol], n = 12) in microscale volumes from 2 to 8 µL (Fig. 4b). Though variation in Am of [18F]fallypride at the macroscale was not systematically measured as a function of reaction volume, we noted that Am for the synthesis at 0.5 mL scale was significantly lower (i.e., 0.9 ± 0.05 Ci/µmol [33 ± 2 GBq/µmol]; n = 3) than the chip results. These data demonstrate that reagents are the dominant source of 19F-contamination at the macroscale, but are negligible relative to other sources at the microscale.

Though not related to molar activity, it is interesting to note that the decay-corrected radiochemical conversion of [18F]fluoride into 18F-labeled tracer in macro- and microscales (as measured by radio-TLC) were similar, and remained relatively constant across all volumes tested. Conversion was 67 ± 9% (n = 11) at the macroscale and 54 ± 14% (n = 12) at the microscale for [18F]FNB, and 77 ± 1% (n = 3) and 71 ± 2% (n = 12), respectively, for [18F]fallypride. Despite varying reaction volume at each synthesis scale, the conversion exhibited relatively little variation, perhaps due to the use of identical reagent concentrations.

Increasing the amount of the radioisotope is known to increase the molar activity at the macroscale, which is the rationale for using high starting radioactivities when producing a tracer with high Am29, 30. We compared the molar activity for both [18F]FNB and [18F]fallypride syntheses at the macroscale, starting from different radioactivities (8–360 mCi [0.3–13.3 GBq]) while maintaining a constant reaction volume (0.5 mL). A linear relationship between Am and starting activity for both tracers was observed (Fig. 4c). Comparative experiments were then conducted in microscale on-chip syntheses with fixed reaction volume (4 µL). Interestingly, Am was relatively constant for both tracers (~20–25 Ci/µmol [740–930 GBq/µmol]), despite a wide range of starting radioactivities used (25–525 mCi [0.9–19.4 GBq]) (Fig. 4c).

An interpretation for these results is depicted in Fig. 5. Because the starting radioactivity was varied by adding different volumes of the [18F]fluoride solution from the cyclotron, increasing the starting radioactivity therefore also increases the cyclotron source of 19F-contamination. At the macroscale, we concluded above that reagents are the dominant source of 19F-contamination; thus, even as starting activity is increased, we expect the associated increase in 19F-contamination from the cyclotron to be negligible in comparison. Equation 1 then becomes Am ≈ Ammax [18FC/(18FC + 19FS)] ≈ Ammax (18FC/19FS). (The second approximation is made since the number of fluorine-19 typical far outnumbers the number of fluorine-18 ions.) This equation describes a linear dependence of molar activity on the amount of [18F]fluoride, as observed in our study. On the other hand, at the microscale, we previously concluded in the previous section that the reagents are a negligible source of 19F-contamination. As more radioactivity is added, we thus expect the dominant source of [19F]fluoride (cyclotron) to increase in proportion with [18F]fluoride. Equation 1 becomes Am ≈ Ammax [18FC/(18FC + 19FC)] ≈ Ammax (18FC/19FC). This expression is a constant for a given batch of the isotope, as we observed.

### Effect of fluorinated materials

Radiolysis of fluorinated materials, such as Teflon, are known to lead to reduction in molar activity at the macroscale37. We investigated the possibility that the Teflon coating of the EWOD chips, in contact with reagents before and during the fluorination process, could adversely impact Am in the microscale synthesis. Surprisingly, experiments assessing the impact of Teflon exposure to the initial [18F] fluoride solution as a function of surface area, duration, and radioactivity showed negligible impact on the Am of the resulting tracers (Supplementary Figure 1, Supplementary Discussion).

The above trends suggest that, in microfluidic 18F-radiosynthesis, reagents and Teflon contact do not have an appreciable effect on Am compared to cyclotron sources of 19F-contamination. Microdroplet-based radiosynthesis therefore provides a route, under the radiochemist’s control, for maximizing molar activity of PET tracers from a given batch of the radioisotope.

### Effect of molar activity on in vivo imaging

While a few reports have compared the impact of low vs high molar activity in small animal imaging20, 47,48,49, this subject does not appear to have been extensively studied. In general, higher molar activity is expected to be important for imaging low density targets by reducing competition for binding sites with the non-radioactive form of the tracer. On the other hand, some tracers will benefit from lower Am such as those with a substantial “sink” outside of the target tissue (e.g., non-specific binding or metabolism)27. In general, many factors could affect imaging and optimal molar activity may have to be evaluated experimentally for particular applications and having high Am facilitates these evaluations.

We empirically studied the impact of molar activity on in vivo imaging of the saturable dopamine D2/D3 system with [18F]fallypride. In two replicate experiments, a panel of mice were injected with the same amount of radioactivity, but each with a different Am. Note that in this section, Am values are corrected to the time of injection and not EOB. Static scans after 60 min uptake are shown in Fig. 6. Substantial differences were observed in the images depending on Am. In the first study (Fig. 6a), Am ≥ 0.49 Ci/µmol [18 GBq/µmol] resulted in significant image contrast between striatum (region with high density of D2/D3 receptors) and cerebellum (where no binding is expected), but the striata remained undetectable at Am = 0.059 Ci/µmol [2.2 GBq/µmol]. Similarly in the second study (Fig. 6b), significant image contrast is seen at Am ≥ 0.96 Ci/µmol [36 GBq/µmol], whereas the striata was undetectable at Am = 0.009 Ci/µmol [0.33 GBq/µmol]. These results, and calculated occupancies (see Supplementary Table 1, Supplementary Methods) are consistent with the report of Fischer et al. that this receptor system in mice is expected to starting showing significant (>10%) occupancy for injected amounts >4 nmol/kg50.

Dynamic images revealed significant kinetic differences related to molar activity (see Supplementary Figures 2 and 3). For Am ≥ 0.49 Ci/µmol [18 GBq/µmol], [18F]fallypride retention in the striatum (Supplementary Figures 2a, 3a) rapidly increased from the beginning of the scan until ~200 s. Retention remained nearly constant thereafter for Am ≥ 0.96 Ci/µmol [36 GBq/µmol], with signal intensity correlating with Am. For Am ≤ 0.49 Ci/µmol [18 GBq/µmol], significant washout occurred after initial uptake, resulting in relatively low signal by the end of the 1 h scan, with lower retention for lower Am. For the lowest Am in each study, the initial rapid uptake was not observed (did not occur or was shorter than the frame duration). In the cerebellum, an initially high signal rapidly decreased (Supplementary Figures 2b, 3b), the decrease being faster for lower Am. The striatum:cerebellum ratio increased over time and was generally higher for higher molar activity, though midrange Am values (0.49–2.0 Ci/µmol [18–74 GBq/µmol]) seemed to provide the greatest ratio (Supplementary Figures 2c, 3c). While additional studies involving more replicates and increased coverage of Am values are needed to develop a better understanding of the influence of Am, it is clear from these results that, at least for [18F]fallypride, molar activity can have substantial impact on the quantitative analysis and qualitative interpretation of PET images.

## Discussion

Our study showed that macroscale and microscale radiosynthesis had marked differences in the effect of several parameters on molar activity of 18F-labeled PET tracers. The data suggest that the advantage of microscale synthesis is the minimization of 19F-contamination in reagents. By reducing reaction volume by 2–3 orders of magnitude, sources of contamination in the synthesis became negligible in comparison to contamination already present in the initial [18F]fluoride solution from the cyclotron. Microdroplet synthesis thus provides the radiochemist a tool for achieving the highest possible molar activity. The consistency of microscale Am values over a wide range of conditions suggests that Am likely matches Amisotope. Indeed, values are consistent with measurements over several years in our cyclotron facility suggesting Amisotope is ~20 Ci/µmol [740 GBq/µmol]. Since other facilities have reported similar values17, we expect comparable results could be achieved using [18F]fluoride from other cyclotrons.

This ability to reliably synthesize tracers with high Am provides important advantages in the screening and development of novel tracers, especially at early stages when very little may be known about toxicity, pharmacokinetics, target abundance, etc. Since the impact of Am on qualitative and quantitative imaging can be significant, as was seen with [18F]fallypride, it may be desirable to always err on the side of very high Am to avoid potential pharmacologic response or target occupancy issues (which could lead to false negative results). Alternatively, it may be useful to be able to produce a small batch of a new tracer with very high Am, aliquot several portions and adjust each to a different (lower) Am by addition of non-radioactive tracer, and then studying the effect in vivo to determine the optimal range of molar activity for the intended application. High Am also enables increases in throughput of animal experiments by enabling the batch of tracer to be used over a longer period of time and still have sufficient molar activity at the time of injection.

We suspect the ability of microscale radiosynthesis to achieve high molar activity is not limited to the model compounds chosen here, and can likely be generalized to most 18F-labeled tracers44 and potentially also to tracers labeled with other isotopes (e.g., radiometals). In addition to reducing isotopic contamination, microvolume radiochemistry may also provide a simple route to reduce the absolute amount of precursor used, while maintaining a high concentration of reagent during the radiolabeling reaction to ensure a fast reaction rate. This will improve Am for isotopic exchange reactions33 and apparent Am for labeling of larger molecules such as proteins, where labeled and unlabeled species cannot be separated during purification. Furthermore, reduction in precursor would likely enable simpler purification processes.

## Methods

### Reagents

2,3-dimethyl-2-butanol (98%), anhydrous acetonitrile (MeCN; HPLC grade, >99.9%), anhydrous dimethylsulfoxide (DMSO; HPLC grade, 99.9%), hexane (HPLC grade, >97%), ethyl acetate (HPLC grade, >99.8%), ammonium formate (HCOOHNH4) (HPLC grade, >99%), triethylamine (TEA; >99%), 1,4-dinitrobenzene (98%), 1-fluoro-4-nitrobenzene (99%), ethanol (EtOH; 200 proof), methanol (MeOH; HPLC grade, >99.9%), Fluorinert® FC40 and K2CO3 (>99%) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). 2,3-dimethoxy-5-[3-[[(4-methylphenyl)sulfoniyl]oxy]propyl]-N-[[1-(2-propenyl-2-pyrrolidinyl)methyl] (tosyl-Fallypride precursor) (>90%), Fallypride standard compound (>95%), tetrabutylammonium bicarbonate (TBAHCO3) 0.075 M aq. solution stabilized with ethanol, and Cryptand-222 (K-222; >99%) were purchased from ABX Advanced Biochemical Compounds (Radeberg, Germany). Teflon® AF2400 solution (6%) for preparation of microfluidic chips was purchased from DuPont (USA). All chemicals were used without further purification.

No-carrier-added [18F]fluoride ion was obtained from the UCLA Biomedical Cyclotron Facility, where it was produced by irradiation of 85% 18O-enriched water with an 11 MeV proton beam using an RDS-112 cyclotron (Siemens Medical Solutions, Knoxville, TN). Typically, a ~1 Ci [37 GBq] bombardment was performed and the resulting [18F]fluoride/[18O]water solution was obtained in a volume of ~1.0 mL. The delivery time from the production site to our facility was sufficient to ensure any nitrogen-13 produced in the bombardment had decayed by the time of use.

### Analytical methods

Radioactivity was determined using a calibrated ion chamber (CRC-25 PET, Capintec).

Fluorination efficiency (conversion efficiency) was analyzed via radio thin layer chromatography (radio-TLC). A sample was spotted onto a TLC plate (IB-F silica gel plates, BakerFlex). After developing in the mobile phase, the plate was dried and scanned by a radio-TLC scanner (MiniGITA star, Raytest USA, Inc, Wilmington, NC). Mobile phase for analysis of [18F]fallypride was 60:40 (v/v) MeCN:25 mM ammonium formate with 1%TEA, and the mobile phase for [18F]FNB was 60:40 (v/v) MeCN:H2O. Rf values for unreacted [18F]fluoride, [18F]fallypride, and [18F]FNB were 0.0, ~0.75, and ~0.80, respectively.

Analytical HPLC analysis for determination of purity and molar activity was performed on a Smartline HPLC system (Knauer, Germany), equipped with a degasser (Model 5050), pump (Model 1000), Phenomenex Luna reversed-phase C-18 column (250 × 4.6 mm) equipped with guard column (SecurityGuard C-18, Phenomenex, Torrance, CA), a variable wavelength UV detector (Model 2500) and a radiometric detector (Bioscan B-FC-4000, Bioscan Inc., Washington DC, USA). For [18F]fallypride samples, the mobile phase used was 60:40 (v/v) MeCN: 25 mM ammonium formate with 1% v/v TEA at a flow rate of 1.5 mL/min. The retention time of Fallypride was ~8.0 min. For [18F]FNB, the mobile phase used was 55:45 (v/v) MeCN:H2O at a flow rate of 1 mL/min. The retention time of [18F]FNB was ~ 25 min.

### Determination of molar activity

From the UV detector chromatogram, the area under the curve (AUC) of the peak corresponding to the tracer is determined. Using the appropriate calibration curve, this value is converted to the number of moles of compound injected. Finally, the determined radioactivity value was divided by molar amount of the tracer to obtain the molar activity.

To avoid introducing Am variations caused by different times of use of the radioactivity, which would confound the phenomena we were trying to study, all Am values were decay-corrected to the end of bombardment (EOB). For imaging purposes, we recognize that one would generally be interested in the molar activity at the time of injection. Assuming the synthesis is started at EOB (to maximize Am and the available amount of radioactivity), then Am at the time of injection would typically be 43% lower than the decay-corrected values we report (estimating synthesis and formulation time of ~90 min).

To build the calibration curves (Supplementary Figure 4), stock solutions of fallypride and FNB reference standards were injected into HPLC, resulting in chromatograms with a single peak. The amount injected was varied by using different injection volumes. The AUC of each peak was determined, and the AUCs were plotted as a function of injected amount of each compound. A linear least squares fit was then performed for each compound (R2 = 0.9906 for fallypride and R2 = 0.9986 for FNB). The range of concentrations was chosen to cover the expected range of masses in samples.

Theoretically, chlorination of the precursor is also possible by chloride ions that may be present as contamination in the initial [18F]fluoride solution originating from the cyclotron or might be present in reagents. To ensure that UV peaks represent only the fluorinated form, we compared the retention times of the standard solutions of the fluoro- and chloro-derivatives (Supplementary Figure 5). These forms will well-separated, ruling out the possibility that the chloro-derivatives could affect the molar activity measurements.

Microscale radiosynthesis was carried out in droplets on model EWOD chips consisting of Teflon AF-coated glass slides (Fig. 2). These simplified chips are significantly less expensive than EWOD chips, but have similar thermal properties and identical materials in contact with the reaction mixture. Fabrication of chips was similar to previously reported methods40. The chips were made from glass pieces (25 mm × 25 mm) cut from standard microscope slides (Fisherbrand Plain Glass Microscope Slides). After cleaning with MeOH, acetone, and DI water, the glass pieces were spin-coated with a 1% Teflon® AF2400 solution (prepared by diluting the 6% solution with Fluorinert® FC40). The coating protocol consisted of three steps: 5 s at 500 rpm (100 rpm/s ramping rate), 30 s at 1000 rpm (100 rpm/s ramping rate), and then 0 rpm (500 rpm/s ramping rate). Subsequently, Teflon-coated glass slides were placed in an open Pyrex® Petri dish, and underwent baking on a hotplate at 150, 180, and 350 °C for 10, 10, and 60 min, respectively, to remove solvent and anneal the Teflon layer.

To control temperature, the chip was affixed on top of a small aluminum heating block (1 × 1 × 1 cm) containing a thermistor for temperature measurement. The heating block was in turn was placed on top of a thermoelectric (Peltier) heating/cooling element. Temperature was maintained using a thermoelectric controller (5R7-001, McShane Inc., Medina, OH, USA). Thermal paste (OmegaTherm 201, Omega Engineering, Inc., Norwalk, CT, USA) was applied between components to ensure good thermal contact. Reagents were loaded onto the chip via micropipette.

The microscale synthesis of [18F]fallypride was adapted from our previous reports43, 45. A [18F]fluoride/base stock solution was prepared by mixing 0.075 M TBAHCO3 (5 µL) with [18F]fluoride/[18O]H2O (100 µL; ~100 mCi [3.7 GBq]), corresponding to an initial radioactivity concentration of ~1.0 mCi/µL [37 MBq/µL]. A precursor stock solution was prepared by dissolving tosyl-fallypride (4 mg) in 1:1 v/v MeCN/thexyl alcohol (100 µL).

A droplet of the [18F]fluoride/base stock solution (8 μL) and a droplet of MeCN (8 μL) were first deposited on a new chip. To achieve a total radioactivity of ~20 mCi [0.74 GBq], additional [18F]fluoride/[18O]water (12 µL) and MeCN (12 μL) were added. The chip was heated to 105 °C (actual ramp time ~140 s) to evaporate the solvent, leaving a dried residue of the [18F]TBAF complex. Residual water was removed by adding another droplet of MeCN (8 µL) and evaporating at 105 °C for 1 min. Note that the dried residue covers an area much smaller than the original droplet size. A droplet of precursor solution (4 µL) was added to the residue site, and the reaction droplet was then sandwiched by placing a separate Teflon-glass plate as a “cover plate” on top of the droplet. The gap height was set to ~150 µm by using small pieces of double-sided Scotch 3 M tape spacers attached to the edges of the second glass plate. The fluorination reaction was performed by heating to 105 °C for 7 min. To collect the crude product for analysis, the top chip was removed and a droplet of MeOH (20 µL) was added to each chip, briefly mixed, and collected with a pipette. This process was repeated an additional time, resulting in a total collected volume of ~80 µL.

When used for imaging, the crude [18F]fallypride was purified using the analytical-scale HPLC method described above, except with flow rate of 1 mL/min (giving retention time of ~12 min). The collected eluate was subsequently evaporated in a V-vial using an oil bath (110 °C), redissolved in a 3% v/v EtOH:saline mixture (1 mL) and passed through 0.22 µm sterilizing filter (Anotop® 10 Plus, Whatman) into a sterile empty vial. The final activity in the product vial was ~10 mCi [0.37 GBq].

The microscale synthesis of [18F]FNB was performed similarly by adapting the macroscale conditions reported by Collins et al.53. A base stock solution was first prepared by dissolving K2CO3 (2.5 mg) and K-222 (30 mg) in water (350 µL). A 50 µL aliquot was mixed with 50 µL of [18F]fluoride/[18O]H2O (~50 mCi [1.9 GBq]) to prepare the [18F]fluoride/base stock solution, corresponding to a radioactivity concentration of ~0.5 mCi/µL [~19 MBq/µL]. A precursor stock solution was prepared by mixing 1,4-dinitrobenzene (2.5 mg) in 145 µL of DMSO.

A droplet of the [18F]fluoride/base solution (8 μL) and a droplet of MeCN (8 μL) were added onto a new chip, followed by additional droplets of [18F]fluoride/[18O]water (16 µL) and MeCN (16 µL) to yield a total radioactivity of ~20 mCi [0.74 GBq]. The chip was heated to 105 °C to evaporate the solvent. After cooling, an additional droplet of MeCN (8 µL) was added and evaporated at 105 °C for 1 min to azeotropically dry the [18F]KF/K-222 complex. The precursor solution droplet (4 µL) was then added and a cover chip installed. The chip was heated to 130 °C (actual ramp time ~210 s) for 5 min to perform the fluorination reaction. After cooling to 25 °C (actual ramp time ~170 s), the crude product was collected in MeOH in the same fashion as for [18F]fallypride.

Macroscale radiosyntheses were performed using the ELIXYS automated radiosynthesizer (Sofie Biosciences, Inc., Culver City, CA, USA)54, 55.

The macroscale synthesis of [18F]fallypride was adapted from our previous report55. A base stock solution was prepared by mixing 0.075 M TBAHCO3 (600 µL) and MeCN (1 mL). A precursor stock solution was prepared by dissolving tosyl-fallypride (4 mg) in 1:1 v/v MeCH:thexyl alcohol (0.5 mL).

First, the reactor vial was loaded with a solution of [18F]fluoride/[18O]H2O (20 µL; ~20 mCi [0.74 GBq]) and base stock solution (1.6 mL), and subsequently dried at 110 °C until almost dry (2.5 min). Anhydrous MeCN (1 mL) was then added to reaction vial to azeotropically remove water from the solution by heating at 110 °C until complete dryness (1.5 min). This step was repeated an additional time to ensure sufficient removal of water. Next, precursor solution (0.5 mL) was added into reactor vial containing the dried [18F]TBAF complex. The fluorination reaction was performed at 110 °C for 7 min, and the resulting crude mixture was analyzed.

The macroscale synthesis of [18F]FNB was adapted from the procedure of Collins et al.53. Base stock solution was prepared by dissolving K2CO3 (12 mg) in H2O (1200 µL). Cryptand stock solution was prepared by dissolving K-222 (120 mg) in MeCN (5400 µL). A precursor stock solution was prepared by mixing 1,4-dinitrobenzene (2 mg) in DMSO (1 mL).

First, the reactor vial was loaded with solutions of [18F]fluoride/[18O]H2O (20 µL; ~20 mCi [0.74 GBq]), base stock solution (100 µL), cryptand stock solution (900 µL), and acetonitrile (1 mL). Drying was performed in an identical fashion as for [18F]fallypride. Next, precursor stock solution (1 mL) was added into reactor vial containing the dried [18F]KF/K-222 complex. The fluorination reaction was performed at 130 °C for 7 min, and then the reactor was cooled for analysis of the crude product.

### Molar activity comparisons

The effect of several different synthesis factors on Am was investigated via a systematic series of experiments. Sets of experiments were always performed from a single batch of [18F]fluoride/[18O]water to eliminate potential day-to-day variation (e.g., of Amisotope) as a variable. In addition, all the chips used throughout the whole day were taken from the same fabrication batch, and reagents and solvents used were taken from the same product batch.

Note that processing of [18F]fluoride via an anion exchange cartridge (e.g., quaternary methylammonium, QMA) was not performed in these studies, as the 19F-contamination introduced in typical preconditioning processes39 could confound the measurements. Detailed investigation of this step and optimization for high Am will be performed in the future.

### Effect of reaction volume on molar activity

To understand the influence of reagent amounts on [19F]fluoride contamination, experiments were performed in which the volume of [18F]fluoride/[18O]H2O was held constant, while the volume of reagent stock solutions (base, phase transfer catalyst, and precursor) were varied proportionally. The influence of reaction volume on Am was studied at both the microscale ([18F]fallypride and [18F]FNB) and macroscale ([18F]FNB). In the macroscale case (Fig. 3a), identical amounts of [18F]fluoride/[18O]water (30 µL; ~30 mCi [1.1 GBq]) were loaded into each of four empty reaction vials. The amounts of all other reagents were scaled proportionally to ensure consistent concentrations and reagent ratios (see Supplementary Table 2). Different volumes of base/phase transfer catalyst stock solutions were added to each vial during [18F]fluoride dyring, and corresponding volumes of precursor stock solution were added to each vial during the fluorination reaction. Reactions were performed in a back-to-back fashion in the ELIXYS radiosynthesizer using multiple reactor positions, and multiple reagent delivery positions for each reactor. Am was compared for the different reactions. The set of four reactions was repeated three times (on different days). The microscale syntheses were performed in an analogous fashion (Fig. 3c) at slightly smaller radioactivity scale (~20 mCi [0.74 GBq]). Different amounts [18F]fluoride/base mixture, MeCN, and [18F]fluoride/[18O]water (see Supplementary Table 3) were loaded onto each of four chips to achieve an analogous comparison (i.e., identical amounts of [18F]fluoride/[18O]H2O but varied amount of other reagents), and the syntheses was carried out at described above. Am of the crude reaction mixtures from different chips were compared. The set of four reactions was repeated three times (on different days) for each of the model compounds.

### Effect of starting radioactivity on molar activity

The effect of starting radioactivity on Am was explored at both the macroscale and microscale for both model compounds. For the macroscale case (Fig. 3b), different volumes of [18F]fluoride/[18O]water were initially loaded into the reaction vials to achieve different starting radioactivity. [18F]fallypride synthesis was carried out as described above, and [18F]FNB synthesis was carried out as described above except all reagent volumes were reduced 2× (i.e., to match 500 µL fluorination volume). For the microscale experiments (Fig. 3d), synthesis of the two compounds was performed as described above (i.e., with 4 µL fluorination reaction volume), except varied volumes of additional [18F]fluoride/[18O]H2O were added after the 8 µL of [18F]fluoride/base solution to achieve different starting radioactivities. Note that when the total volume was >200 µL, the capacity of the chip was exceeded, and it was necessary to load a portion at a time (up to 200 µL) and dry it, before adding the next portion.

### Effects of Teflon surface on molar activity

Since it is known that radiolysis of bulk Teflon can introduce [19F]fluoride ion into the reaction mixture in proportion to the time of exposure, surface area of exposure, and amount of radioactivity37, we also performed several experiments to evaluate if there is any influence of the Teflon coating on molar activity of a probe synthesized using the microchip (see Supplementary Methods).

### In vivo imaging and analysis

Beginning with a high Am production of [18F]fallypride using a microfluidic chip, four formulations with different Am (at time of injection) were prepared by relying both on radioactive decay and spiking with non-radioactive standard ([19F]Fallypride). Am of each formulation was separately measured via analytical radio-HPLC just prior to injection. Static and dynamic imaging was performed to determine the effects of Am on imaging.

All in vivo imaging studies were conducted in accordance with UCLA Animal Research Committee-approved protocols and guidelines. Healthy female BALB/c mice (8 weeks old) were anesthetized with 2% isoflurane in oxygen, catheterized via tail vein, and injected with ~100 µCi [~3.7 MBq] [18F]fallypride via catheter. Mice underwent either 10-min static PET scans after a 60 min uptake period under anesthesia or 70-min dynamic PET scans (Inveon microPET, Siemens Medical Solutions USA Inc.). Images were processed with 3D histogramming and reconstruction with a zoom factor of 2.1 using 3D-OSEM with two iterations followed by MAP with 18 iterations. All PET procedures were followed with CT scans (CrumpCAT microCT56) for anatomical co-registration. PET/CT images were analyzed using AMIDE version 1.0.557. Regions of interest (ROIs) were drawn for the left striatum, right striatum, and cerebellum to calculate the ratio of specific (striatal) to non-specific (cerebellum) [18F]fallypride binding.

### Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

## References

1. 1.

Vallabhajosula, S., Solnes, L. & Vallabhajosula, B. A broad overview of positron emission tomography radiopharmaceuticals and clinical applications: what is new? Semin. Nucl. Med. 41, 246–264 (2011).

2. 2.

Ametamey, S. M., Honer, M. & Schubiger, P. A. Molecular imaging with PET. Chem. Rev. 108, 1501–1516 (2008).

3. 3.

Hargreaves, R. et al. Optimizing central nervous system drug development using molecular imaging. Clin. Pharmacol. Ther. 98, 47–60 (2015).

4. 4.

Willmann, J. K., van Bruggen, N., Dinkelborg, L. M. & Gambhir, S. S. Molecular imaging in drug development. Nat. Rev. Drug Discov. 7, 591–607 (2008).

5. 5.

Matthews, P. M., Rabiner, E. A., Passchier, J. & Gunn, R. N. Positron emission tomography molecular imaging for drug development. Br. J. Clin. Pharmacol. 73, 175–186 (2012).

6. 6.

Zhu, L., Ploessl, K. & Kung, H. F. PET/SPECT imaging agents for neurodegenerative diseases. Chem. Soc. Rev. 43, 6683–6691 (2014).

7. 7.

Honer, M., Gobbi, L., Martarello, L. & Comley, R. A. Radioligand development for molecular imaging of the central nervous system with positron emission tomography. Drug Discov. Today 19, 1936–1944 (2014).

8. 8.

Phelps, M. E. Molecular imaging with positron emission tomography. Annu. Rev. Nucl. Part. Sci. 52, 303–338 (2002).

9. 9.

Phelps, M. E. Positron emission tomography provides molecular imaging of biological processes. Proc. Natl Acad. Sci. USA 97, 9226–9233 (2000).

10. 10.

11. 11.

Alauddin, M. M. Positron emission tomography (PET) imaging with 18F-based radiotracers. Am. J. Nucl. Med. Mol. Imaging 2, 55–76 (2012).

12. 12.

Chyng-Yann, Shiue, Fowler, Joanna S., Wolf, Alfred P., Watanabe, Masazumi & Arnett, Carroll D. Synthesis and specific activity determinations of no-carrier-added fluorine-18-labeled neuroleptic drugs. J. Nucl. Med. 26, 181–186 (1985).

13. 13.

Gómez-Vallejo, V., Gaja, V., Koziorowski, J. & Llop, J. in Positron Emission Tomography - Current Clinical and Research Aspects (ed. Hsieh, C.-H.) (InTech, Rijeka, Croatia, 2012).

14. 14.

Eckelman, W. C., Bonardi, M. & Volkert, W. A. True radiotracers: are we approaching theoretical specific activity with Tc-99m and I-123? Nucl. Med. Biol. 35, 523–527 (2008).

15. 15.

Cai, L., Lu, S. & Pike, V. W. Chemistry with [18F]fluoride ion. Eur. J. Org. Chem. 2008, 2853–2873 (2008).

16. 16.

Liu, Z. et al. Stoichiometric leverage: rapid 18F-aryltrifluoroborate radiosynthesis at high specific activity for click conjugation. Angew. Chem. Int. Ed. 52, 2303–2307 (2013).

17. 17.

Liu, Z. et al. Kit-like 18F-labeling of RGD-19F-Arytrifluroborate in high yield and at extraordinarily high specific activity with preliminary in vivo tumor imaging. Nucl. Med. Biol. 40, 841–849 (2013).

18. 18.

Innis, R. B. et al. Consensus nomenclature for in vivo imaging of reversibly binding radioligands. J. Cereb. Blood Flow Metab. 27, 1533–1539 (2007).

19. 19.

Hume, S. P., Gunn, R. N. & Jones, T. Pharmacological constraints associated with positron emission tomographic scanning of small laboratory animals. Eur. J. Nucl. Med. Mol. Imaging 25, 173–176 (1998).

20. 20.

Jagoda, E. M., Vaquero, J. J., Seidel, J., Green, M. V. & Eckelman, W. C. Experiment assessment of mass effects in the rat: implications for small animal PET imaging. Nucl. Med. Biol. 31, 771–779 (2004).

21. 21.

Madsen, K., Marner, L., Haahr, M., Gillings, N. & Knudsen, G. M. Mass dose effects and in vivo affinity in brain PET receptor studies — a study of cerebral 5-HT4 receptor binding with [11C]SB207145. Nucl. Med. Biol. 38, 1085–1091 (2011).

22. 22.

Bergström, M., Grahnén, A. & Långström, B. Positron emission tomography microdosing: a new concept with application in tracer and early clinical drug development. Eur. J. Clin. Pharmacol. 59, 357–366 (2003).

23. 23.

Lappin, G. & Garner, R. C. Big physics, small doses: the use of AMS and PET in human microdosing of development drugs. Nat. Rev. Drug Discov. 2, 233–240 (2003).

24. 24.

Rani, Pu & Naidu, M. U. Phase 0 - microdosing strategy in clinical trials. Indian J. Pharmacol. 40, 240–242 (2008).

25. 25.

Agdeppa, E. & Spilker, M. A review of imaging agent development. AAPS J. 11, 286–299 (2009).

26. 26.

Liu, Z. et al. Rapid, one-step, high yielding 18F-labeling of an aryltrifluoroborate bioconjugate by isotope exchange at very high specific activity. J. Label. Compd. Radiopharm. 55, 491–496 (2012).

27. 27.

Kung, M.-P. & Kung, H. F. Mass effect of injected dose in small rodent imaging by SPECT and PET. Nucl. Med. Biol. 32, 673–678 (2005).

28. 28.

Lapi, S. E. & Welch, M. J. A historical perspective on the specific activity of radiopharmaceuticals: what have we learned in the 35 years of the ISRC? Nucl. Med. Biol. 40, 314–320 (2013).

29. 29.

Libert, L. C. et al. Production at the Curie level of no-carrier-added 6-18F-Fluoro-l-Dopa. J. Nucl. Med. 54, 1154–1161 (2013).

30. 30.

Lemaire, C. et al. Automated production at the curie level of no-carrier-added 6-[18F]fluoro- l-dopa and 2-[18F]fluoro- l-tyrosine on a FASTlab synthesizer. J. Label. Compd. Radiopharm. 58, 281–290 (2015).

31. 31.

Ravert, H. T. et al. An improved synthesis of the radiolabeled prostate-specific membrane antigen inhibitor, [18F]DCFPyL. J. Label. Compd. Radiopharm. 59, 439–450 (2016).

32. 32.

Scott, P. J. H. et al. Studies into radiolytic decomposition of fluorine-18 labeled radiopharmaceuticals for positron emission tomography. Appl. Radiat. Isot. 67, 88–94 (2009).

33. 33.

Bernard-Gauthier, V. et al. From unorthodox to established: the current status of 18F-Trifluoroborate- and 18F-SiFA-based radiopharmaceuticals in PET nuclear imaging. Bioconjug. Chem. 27, 267–279 (2016).

34. 34.

Solin, O., Bergman, J., Haaparanta, M. & Reissell, A. Production of 18F from water targets. Specific radioactivity and anionic contaminants. Int. J. Rad. Appl. Instrum. 39, 1065–1071 (1988).

35. 35.

Füchtner, F., Preusche, S., Mäding, P., Zessin, J. & Steinbach, J. Factors affecting the specific activity of [18F]fluoride from a [18O]water target. Nukl. Nucl. Med. 47, 116–119 (2008).

36. 36.

Satyamurthy, N., Amarasekera, B., Alvord, C. W., Barrio, J. R. & Phelps, M. E. Tantalum [18O]water target for the production of [18F]fluoride with high reactivity for the preparation of 2-deoxy-2-[18F]fluoro-D-glucose. Mol. Imaging Biol. 4, 65–70 (2002).

37. 37.

Berridge, M. S., Apana, S. M. & Hersh, J. M. Teflon radiolysis as the major source of carrier in fluorine-18. J. Label. Compd. Radiopharm. 52, 543–548 (2009).

38. 38.

Link, J. M., Shoner, S. C. & Krohn, K. A. Sources of carrier F-19 in F-18 fluoride. AIP Conf. Proc. 1509, 61–65 (2012).

39. 39.

Lu, S., Giamis, A. M. & Pike, V. W. Synthesis of [18F]fallypride in a micro-reactor: rapid optimization and multiple-production in small doses for micro-PET studies. Curr. Radiopharm. 2, 1–13 (2009).

40. 40.

Keng, P. Y. et al. Micro-chemical synthesis of molecular probes on an electronic microfluidic device. Proc. Natl Acad. Sci. USA 109, 690–695 (2012).

41. 41.

Keng, P. Y. & van Dam, R. M. Digital microfluidics: a new paradigm for radiochemistry. Mol. Imaging 14, 579–594 (2015).

42. 42.

Elizarov, A. M. et al. Design and optimization of coin-shaped microreactor chips for PET radiopharmaceutical synthesis. J. Nucl. Med 51, 282–287 (2010).

43. 43.

Javed, M. R. et al. High yield and high specific activity synthesis of [18F]fallypride in a batch microfluidic reactor for micro-PET imaging. Chem. Commun. 50, 1192–1194 (2014).

44. 44.

Javed, M. R. et al. Efficient radiosynthesis of 3′-Deoxy-3′-18F-Fluorothymidine using electrowetting-on-dielectric digital microfluidic chip. J. Nucl. Med. 55, 321–328 (2014).

45. 45.

Chen, S. et al. Radiolabelling diverse positron emission tomography (PET) tracers using a single digital microfluidic reactor chip. Lab. Chip 14, 902–910 (2014).

46. 46.

Keng, P. Y., Sergeev, M. & van Dam, R. M. in Perspectives on Nuclear Medicine for Molecular Diagnosis and Integrated Therapy (eds Kuge, Y., Shiga, T. & Tamaki, N.) 93–111 (Springer, Japan, 2016).

47. 47.

Rominger, A. et al. Validation of the octamouse for simultaneous 18F-fallypride small-animal PET recordings from 8 mice. J. Nucl. Med. 51, 1576–1583 (2010).

48. 48.

Honer, M., Brühlmeier, M., Missimer, J., Schubiger, A. P. & Ametamey, S. M. Dynamic imaging of striatal D2 receptors in mice using quad-HIDAC PET. J. Nucl. Med. 45, 464–470 (2004).

49. 49.

Kuik, W.-J. et al. In vivo biodistribution of no-carrier-added 6-18F-Fluoro-3,4-Dihydroxy-L-Phenylalanine (18F-DOPA), produced by a new nucleophilic substitution approach, compared with carrier-added 18F-DOPA, prepared by conventional electrophilic substitution. J. Nucl. Med. 56, 106–112 (2015).

50. 50.

Fischer, K. et al. Noninvasive nuclear imaging enables the in vivo quantification of striatal dopamine receptor expression and raclopride affinity in mice. J. Nucl. Med. 52, 1133–1141 (2011).

51. 51.

Keng, P. Y., Esterby, M. & van Dam, R. M. in Positron Emission Tomography - Current Clinical and Research Aspects (ed. Hsieh, C.-H.) 153–182 (InTech, Croatia, 2012).

52. 52.

Ory, D. et al. Retention of [18F]fluoride on reversed phase HPLC columns. J. Pharm. Biomed. Anal. 111, 209–214 (2015).

53. 53.

Collins, M., Lasne, M.-C. & Barré, L. Rapid synthesis of N,N′-disubstituted piperazines. Application to the preparation of No carrier added 1-(4-[18F]fluorophenyl)piperazine and of an [18F]-selective ligand of serotoninergic receptors (5HT2 antagonist). J. Chem. Soc., Perkin Trans. 1, 3185–3188 (1992).

54. 54.

Lazari, M. et al. ELIXYS - a fully automated, three-reactor high-pressure radiosynthesizer for development and routine production of diverse PET tracers. EJNMMI Res. 3, 52 (2013).

55. 55.

Lazari, M. et al. Fully automated production of diverse 18f-labeled pet tracers on the ELIXYS multireactor radiosynthesizer without hardware modification. J. Nucl. Med. Technol. 42, 203–210 (2014).

56. 56.

Taschereau, R., Vu, N. T. & Chatziioannou, A. F. Calibration and data standardization of a prototype bench-top preclinical CT. In 2014 IEEE Nuclear Science Symposium and Medical Imaging Conference (NSS/MIC) 1–2 (2014). https://doi.org/10.1109/NSSMIC.2014.7430845.

57. 57.

Loening, A. M. & Gambhir, S. S. AMIDE: a free software tool for multimodality medical image analysis. Mol. Imaging 2, 131–137 (2003).

## Acknowledgements

We thank Dr. Saman Sadeghi, Dr. Umesh Gangadharmath, and the staff of the UCLA Biomedical Cyclotron Facility for generously providing [18F]fluoride for these studies, and Dr. Sung-Cheng (Henry) Huang for valuable discussions about image analysis. This work was supported in part by the National Institute of Biomedical Imaging and Bioengineering (R21 EB015540, T32 EB002101), the National Institute on Aging (R21 AG049918), the National Cancer Institute (U54 CA151819A, i.e., the Caltech/UCLA Nanosystems Biology Cancer Center), the Department of Energy Office of Biological and Environmental Research (DE-SC0005056), and the UCLA Foundation from a donation made by Ralph and Marjorie Crump for the Crump Institute for Molecular imaging. The UCLA Crump Institute’s Preclinical Imaging Technology Center is supported in part by the NIH In Vivo Cellular and Molecular Imaging Centers Grant (P50 CA086306), NIH Cancer Center Support Grant (P30 CA016042), and NIH SPORE Grant (P50 CA092131).

## Author information

### Author notes

• Maxim Sergeev

Present address: Department of Radiology, University Hospitals Cleveland Medical Center, 11000 Euclid Ave., Cleveland, OH, 44106, USA

### Affiliations

1. #### Crump Institute for Molecular Imaging, University of California, Los Angeles (UCLA), 570 Westwood Plaza, Los Angeles, CA, 90095, USA

• Maxim Sergeev
• , Mark Lazari
• , Federica Morgia
• , Jeffrey Collins
• , Olga Sergeeva
• , Michael E. Phelps
• , Jason T. Lee
• , Pei Yuin Keng
•  & R. Michael van Dam
2. #### Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles (UCLA), 650 Charles E. Young Dr. S., Los Angeles, CA, 90095, USA

• Maxim Sergeev
• , Mark Lazari
• , Federica Morgia
• , Jeffrey Collins
• , Olga Sergeeva
• , Michael E. Phelps
• , Jason T. Lee
• , Pei Yuin Keng
•  & R. Michael van Dam
3. #### Department of Bioengineering, Henry Samueli School of Engineering and Applied Science, UCLA, Los Angeles, 90095, CA, USA

• Mark Lazari
•  & R. Michael van Dam
4. #### Physics and Biology in Medicine Interdepartmental Graduate Program, David Geffen School of Medicine, UCLA, Los Angeles, 90095, CA, USA

• Jason Jones
•  & R. Michael van Dam
5. #### Jonsson Comprehensive Cancer Center, UCLA, 8-684 Factor Building, Los Angeles, CA, 90095, USA

• Michael E. Phelps
• , Jason T. Lee
• , Pei Yuin Keng
•  & R. Michael van Dam

### Contributions

M.S., M.L., J.T.L., P.Y.K., and R.M.V. conceived and designed the experiments; M.S., M.L., F.M., J.C., M.R.J., O.S., and J.J. performed the experiments; M.S., M.L., F.M., J.T.L., P.Y.K., and R.M.V. analyzed data; M.S., M.E.P., J.T.L., P.Y.K., and R.M.V. wrote the manuscript with input from all authors.

### Competing interests

Drs. van Dam and Phelps are founders of Sofie Biosciences, Inc., Dr. Phelps is a board member of Sofie Biosciences, and Drs. van Dam and Keng are consultants to Sofie Biosciences. The Regents of the University of California have licensed technology to Sofie Biosciences that was invented by Drs. van Dam, Keng, and Lazari, and have taken equity in Sofie Biosciences as part of the licensing transaction.

### Corresponding authors

Correspondence to Pei Yuin Keng or R. Michael van Dam.