Cerenkov Radiation Energy Transfer (CRET) Imaging: A Novel Method for Optical Imaging of PET Isotopes in Biological Systems

Positron emission tomography (PET) allows sensitive, non-invasive analysis of the distribution of radiopharmaceutical tracers labeled with positron (β(+))-emitting radionuclides in small animals and humans. Upon β(+) decay, the initial velocity of high-energy β(+) particles can momentarily exceed the speed of light in tissue, producing Cerenkov radiation that is detectable by optical imaging, but is highly absorbed in living organisms.To improve optical imaging of Cerenkov radiation in biological systems, we demonstrate that Cerenkov radiation from decay of the PET isotopes (64)Cu and (18)F can be spectrally coupled by energy transfer to high Stokes-shift quantum nanoparticles (Qtracker705) to produce highly red-shifted photonic emissions. Efficient energy transfer was not detected with (99m)Tc, a predominantly γ-emitting isotope. Similar to bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), herein we define the Cerenkov radiation energy transfer (CRET) ratio as the normalized quotient of light detected within a spectral window centered on the fluorophore emission divided by light detected within a spectral window of the Cerenkov radiation emission to quantify imaging signals. Optical images of solutions containing Qtracker705 nanoparticles and [(18)F]FDG showed CRET ratios in vitro as high as 8.8±1.1, while images of mice with subcutaneous pseudotumors impregnated with Qtracker705 following intravenous injection of [(18)F]FDG showed CRET ratios in vivo as high as 3.5±0.3.Quantitative CRET imaging may afford a variety of novel optical imaging applications and activation strategies for PET radiopharmaceuticals and other isotopes in biomaterials, tissues and live animals

Molecular Imaging of Pulmonary Disease In Vivo

Characterization and noninvasive measurement of molecular pathways and biochemistry in living cells, animal models, and humans at the cellular and molecular level is now possible using remote imaging detectors. Positron and single photon emission tomography scanners, highly sensitive cameras for bioluminescence and fluorescence imaging, as well as high-magnetic-field magnetic resonance imaging scanners, can be used to study such diverse processes as signal transduction, receptor density and function, host response to pathogens, cell trafficking, and gene transfer. In many cases, images from more than one modality can be fused, allowing structure–function and multifunction relationships to be studied on a tissue-restricted or regional basis. “Molecular imaging” holds enormous potential for elucidating the molecular mechanisms of pulmonary disease and therapeutic response in intact animal models and humans

CRET imaging of pseudotumor phantoms in live animals <i>in vivo</i>.

<p>(<b>A</b>) Subcutaneous pseudotumors of 500 nM Qtracker705-impregnated Matrigel (closed arrow) and PBS-impregnated Matrigel (open arrow) in opposing flanks of <i>nu/nu</i> mice were imaged with an IVIS 100 using open, <510 nm (blue), 500–570 nm (green), and >590 nm (red) filters 30 minutes following tail-vein injection of [¹⁸F]FDG (17.6 MBq; 475 µCi). (<b>B</b>) The calculated CRET image.</p

CRET <i>in vitro</i> was dependent on Qtracker705 nanoparticle concentration.

<p>(<b>A</b>) IVIS 100 images of 96-well assay plates using either a <510 nm filter (left) or a >590 nm filter (right). Note the red “hot pixel” from an annihilation event detected by the CCD camera in one image (left), and the presence of minimally detectable CRET emitted from the wells containing 200 nM Qtracker705, but no [¹⁸F]FDG, due to contaminating radioactive emissions from adjacent wells (right). Qtracker705 nanoparticles show no CRET when imaged in isolation in the absence of [¹⁸F]FDG. (<b>B</b>) Plot of photon flux from either the <510 nm filter (□) or the >590 filter (▪) with increasing concentrations of Qtracker705 nanoparticles. (<b>C</b>) Plot of CRET ratios versus concentration of Qtracker705 nanoparticles (dashed line is a linear fit of the data: y = 0.036x+1.3; R² = 0.897).</p

CRET <i>in vitro</i> was dependent on [¹⁸F]FDG radioactivity.

<p>(<b>A</b>) IVIS 100 images of 96-well assay plates using either a <510 nm filter (left) or a >590 nm filter (right). (<b>B</b>) Plot of photon flux from either the <510 nm filter (□) or the >590 filter (▪) with increasing amounts of [¹⁸F]FDG radioactivity. (<b>C</b>) Plot of CRET ratios versus [¹⁸F]FDG radioactivity.</p

CRET ratios of pseudotumors <i>in vivo</i>.

<p>CRET ratios of pseudotumors <i>in vivo</i>.</p

Spectral analysis.

<p>(<b>A</b>) UV/vis emission spectra of [⁶⁴Cu]CuCl₂ in PBS containing various concentrations of Qtracker705 nanoparticles (Qdots ) demonstrate Cerenkov radiation energy transfer (CRET); (blue) [⁶⁴Cu]CuCl₂ without Qdots, (green) [⁶⁴Cu]CuCl₂ with 49 nM Qdots, (orange) [⁶⁴Cu]CuCl₂ with 222 nM Qdots, (red) [⁶⁴Cu]CuCl₂ with 400 nM Qdots, (black) non-radioactive CuCl₂ without Qdots, (brown) non-radioactive CuCl₂ with 400 nM Qdots, (gray) decayed [⁶⁴Cu]CuCl₂ with 400 nM Qdots. (<b>B</b>) Fluorescence emission spectrum (350 nm excitation) of decayed (>8 half-lives) [⁶⁴Cu]CuCl₂ in PBS containing 400 nM Qtracker705. (<b>C</b>) UV/vis emission spectra of [^99mTc]NaTcO₄ in PBS without (black) and with (red) 400 nM Qtracker705 nanoparticles.</p