We describe a method for enhancing optical detection of PET isotopes in biological systems, termed Cerenkov radiation energy transfer (CRET). Similar in principle to BRET 
and FRET 
, Cerenkov radiation generated by decay of a PET isotope serves as the energy donor and a fluorophore constitutes the energy acceptor. We propose that, in the presence of a fluorophore and Cerenkov radiation, CRET ratios can be calculated as the ratio of light detected within a spectral window centered on the fluorophore emission over light detected within a spectral window of the Cerenkov radiation emission, minus the ratio of light detected in the same filters in the presence of Cerenkov radiation alone (Eq. 1
). As proof of principle, we quantified CRET ratios by imaging the energy transfer of Cerenkov radiation generated from PET isotopes to Qtracker705 nanoparticles both in vitro
and in vivo
Spectra obtained with Qtracker705 in the presence of 64Cu showed an apparent maximum for the energy donor from 400–550 nm for Cerenkov radiation (attributed to inverse wavelength-, refractive index-, depth- and detector-dependent characteristics) and a new acceptor peak centered on 705 nm corresponding to the emission of Qtracker705, consistent with Cerenkov radiation energy transfer. Additionally, we observed with increasing concentrations of Qtracker705 a concomitant decrease in the donor intensity. Such a loss, concordant with formation of the acceptor spectral peak, is similarly observed in FRET. Furthermore, Cerenkov radiation arises from high-energy particles traveling through a medium, inducing transient dipole-moments, while the mechanism of non-radiative (resonance) energy transfer with FRET involves dipole-dipole coupling. However, attributing resonance processes to the CRET phenomena awaits specific mechanistic studies aimed at understanding all possible energy contributions made by decaying PET isotopes in such energy transfer contexts.
Studies in vitro were performed by determining the concentration-dependence of Qtracker705 in the presence of a constant amount of [18F]FDG. We chose nanoparticle concentrations in the nanomolar range, comparable to concentrations that would be feasible in vivo. Within the narrow concentration range of these experiments, we found a quasi-linear increase in CRET with increasing concentrations of Qtracker705. This trend was expected given the sub-optimal excitation that occurs when using such a low-level light source compared to the high molar absorptivity of quantum dot nanoparticles. Additionally, we examined the dose-dependence of [18F]FDG in the presence of a constant amount of Qtracker705. The doses of radiation used in this experiment also were chosen in consideration of applications in vivo, but were limited by high-energy annihilation photons interfering with the CCD detector. We found that CRET ratios increased with radiation dose over the range of doses examined.
Note that the CRET ratio will depend quantitatively on the choice of filters, isotope and fluorophore. Further refinement of CRET imaging is anticipated as optimal filter combinations for various isotopes and fluorophores are discovered and characterized. In this regard, the data in suggest the presence of an isosbestic point at ~680 nm in the CRET spectra of Qtracker705 nanoparticles. Use of narrow bandpass filter sets that include isosbestic points may also enhance quantitative analysis, system calibration, and depth resolution in a variety of experimental conditions that will be explored in the future.
We have demonstrated the imaging applicability of CRET in vivo
using Matrigel pseudotumor phantoms embedded with Qtracker705 or PBS. Following tail-vein injection of [18
F]FDG, we were able to correctly observe the location of Qtracker-loaded pseudotumors as early as five minutes post-injection, a time when [18
F]FDG is widely distributed throughout the blood pool and extracellular spaces. The mean range of positrons emitted by 18
F and 64
Cu is ~0.9 mm (and for very high-energy positron emitters such as 82
Rb is ~7 mm) 
. Thus, [18
F]FDG, in this case the ultimate source of Cerenkov radiation, did not need to be in direct proximity with the nanoparticles to provide adequate energy for CRET, a potential advantage for CRET over FRET or BRET for selected applications. For this reason, there was no need for vessel growth within the pseudotumor for visualization by CRET. Imaging again at 30 minutes post-injection of [18
F]FDG resulted in reduced signal, as would be expected as [18
F]FDG cleared from the blood pool and surface tissues. Conversely, for nanoparticles contained within a vascularized tumor, retention of 18
F]FDG within the tumor cells would provide enhanced proximity and delayed clearance compared to an extracellular source as illustrated in this study. Indeed, the relationship between distance and signal could be quite complex and will need further characterization as CRET imaging is refined. The experiment also was performed using either 200 nM or 500 nM Qtracker705 and, appropriately, the higher concentration yielded a higher CRET ratio, but did not track linearly with the concentration of Qtracker705 in vivo
, as was observed in vitro
. This was likely because of non-linear tissue attenuation of photons in living animals.
As this manuscript was submitted, a report demonstrating the use of low-energy light from γ-emitting 131
I to excite quantum dot nanoparticles in Matrigel phantoms was published independently 
, but with some notable differences. First, in Liu et al. 
, radiotracer was directly admixed into the Matrigel phantoms, rather than injected systemically as in the present study, thereby nominally concentrating the activity by ~3,000-fold (volume of distribution: 2×2×2 mm3
versus a 25 gm mouse
). This favorably allowed use of less radiotracer and quantum nanoparticle material, while reducing background signal, but did not mimic the manner in which this technique would likely be utilized in vivo
. Additionally, 131
I was chosen as the energy donor, which produced emissions dominated by both γ-rays and β−
particles, whereas use of 18
F in the present study, a nearly pure positron emitter, formally confirmed β+
particles as the energy donors and left little ambiguity as to the source of the donor Cerenkov radiation observed. Last, herein we introduce a method for quantitative analysis of the energy transfer process by defining the CRET ratio.
Several optical imaging studies report using Cerenkov radiation as a means of imaging tumors 
, including a recent description of Cerenkov luminescence tomography 
. While the authors were able to correctly identify tumors using this method, by comparison to traditional PET imaging, Cerenkov luminescence imaging alone resulted in substantially lower spatial resolution. This was likely a consequence of the models used for image reconstruction and photonic limitations. Such reconstructions depend on the ability to accurately model photon propagation through heterogeneous tissues. The UV/blue emissions of Cerenkov radiation are highly absorbed in tissues, resulting in relatively short mean pathlengths for these purposes. Thus, one way to overcome the problem of optical diffusion may be to spectrally couple, by energy transfer, the Cerenkov radiation to far-red and near infrared emitting quantum nanoparticles or fluorophores.
While we focus on Cerenkov radiation from β+
particles as the source of excitation energy in this report, in principle, the method should apply to any isotope that emits charged particles that exceed the energy threshold required for Cerenkov radiation in the media (264 keV in water). Thus, β−
particles and α-particles of sufficient energy 
should also enable CRET. It is also possible that other sources of high-energy radiation, such as Bremsstrahlung, radioluminescence from γ-rays, and non-radiative (resonance) energy transfer may also enable energy transfer and the detection of “CRET-like” images. As such, further mechanistic and chemical studies are warranted with other fluorophores and small molecules to determine their relative contributions, strength of signal, and tissue depth-dependence. Quantitative CRET imaging may afford a variety of novel optical imaging applications and activation strategies for studies of PET and other radiopharmaceuticals as well as radiobiology in biomaterials, tissues and live animals.