Cerenkov Luminescence Imaging of Medical
Alessandro Ruggiero*1, Jason P. Holland*2, Jason S. Lewis2,3, and Jan Grimm1,3
1Nuclear Medicine Service, Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, New York;
2Radiochemistry Service, Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, New York;
and3Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York
The development of novel multimodality imaging agents and
techniques represents the current frontier of research in the field
of medical imaging science. However, the combination of
nuclear tomography with optical techniques has yet to be estab-
lished. Here, we report the use of the inherent optical emissions
from the decay of radiopharmaceuticals for Cerenkov lumines-
cence imaging (CLI) of tumors in vivo and correlate the results
with those obtained from concordant immuno-PET studies.
Methods: In vitro phantom studies were used to validate the vis-
ing the positron emitters18F,64Cu,89Zr, and124I; b-emitter131I;
and a-particle emitter225Ac for potential use in CLI. The novel
[DFO]-J591 for immuno-PET of prostate-specific membrane an-
tigen (PSMA) expression was used to coregister and correlate
the CLI signal observed with the immuno-PET images and bio-
distribution studies. Results: Phantom studies confirmed that
Cerenkov radiation can be observed from a range of positron-,
b-, and a-emitting radionuclides using standard optical imaging
devices. The change in light emission intensity versus time was
concordant with radionuclide decay and was also found to cor-
relate linearly with both the activity concentration and the mea-
sured PET signal (percentage injected dose per gram). In vivo
studies conducted in male severe combined immune deficient
mice bearing PSMA-positive, subcutaneous LNCaP tumors
demonstrated that tumor-specific uptake of
could be visualized by both immuno-PET and CLI. Optical and
immuno-PET signal intensities were found to increase over
time from 24 to 96 h, and biodistribution studies were found to
correlate well with both imaging modalities. Conclusion: These
studies represent the first, to our knowledge, quantitative as-
sessment of CLI for measuring radiotracer uptake in vivo.
Manyradionuclides common to both nuclear tomographic imag-
ing and radiotherapy have the potential to be used in CLI. The
value of CLI lies in its ability to image radionuclides that do not
emit either positrons or g-rays and are, thus, unsuitable for use
with current nuclear imaging modalities. Optical imaging of Cer-
enkov radiation emission shows excellent promise as a potential
Key Words: imaging technology; Cerenkov; PET; optical imag-
prostate-specific membrane antigen (PSMA); J591; monoclonal
J Nucl Med 2010; 51:1123–1130
In the field of medical imaging science, the concept of
multimodality is providing the driving force for the
development of the next generation of imaging techniques.
The latest hybrid systems such as PET/CTand PET/MRI are
transforming the clinical management of cancer patients by
consolidating the noninvasive localization and temporal
quantification of changes in tissue function and physiology
available from PET, with the high-resolution anatomic maps
provided by CT or MRI (1,2).
In contrast to the immediate clinical impact of nuclear
tomographic imaging, optical methods such fluorescence-
mediated tomography and bioluminescence imaging have
been largely restricted to use in preclinical models. Reasons
for the limited clinical translation of optical modalities lie in
the inherent limitations imposed by high rates of scattering
and poor tissue penetration at the humanscale. Each of these
limitations leads to increased difficulty in providing the
quantitative analysis of data required for practical applica-
tions in the clinic. As a consequence of these in vivo
limitations, recent advances in the field of optical imaging
have focused on developing methods for imaging micro-
scopic events at the cellular and molecular level.
Endoscopy and surgery could benefit from the translation
of optical imaging techniques to visualize tumor lesions or
metastatic involvement intraoperatively and thereby pro-
vide real-time information to guide surgical resection (3).
However, at present there are no clinically approved
targeted probes for use with targeted fluorescence-reflectance
imaging or fluorescence-mediated tomography. Further tech-
nical and theoretic challenges mean that to date, the
research into developing hybrid systems that combine
nuclear and anatomic methods with optical imaging cam-
eras is limited (1,4). Currently intraoperative methods to
Received Feb. 19, 2010; revision accepted Mar. 19, 2010.
For correspondence or reprints contact: Jan Grimm, Molecular
Pharmacology and Chemistry Program, Memorial Sloan-Kettering
Cancer Center, 1275 York Ave., New York, NY 10065.
*Contributed equally to this work.
COPYRIGHT ª 2010 by the Society of Nuclear Medicine, Inc.
CERENKOV IMAGING OF MEDICAL ISOTOPES • Ruggiero et al.1123
detect radionuclides are limited by the use of hand-held
probes that do not provide any spatial information, whereas
pure optical approaches are limited by the lack of clinically
approved targeted agents. Because 2-dimensional imaging
would require large, expensive, and bulky equipment
unsuitable for an operating suite, no method is currently
available to use the multiplicity of approved radiotracers in
The emission of a continuum of ultraviolet and visible
light from the decay of certain radionuclides in the
condensed phase (now known as the Cerenkov effect)
was first observed in 1926 and was characterized by Pavel
A. Cerenkov in 1934 (5). Later, in 1958—and along with
his colleagues Ilya Frank and Igor Tamm—Cerenkov was
awarded the Nobel Prize in Physics, ‘‘for the discovery and
the interpretation of the Cherenkov effect.’’ Cerenkov
radiation arises when charged particles, such as a b- (b2
or b1) or an a-particle, travel through an optically trans-
parent, insulating material with a velocity that exceeds the
speed of light, c, in the given medium (6). The Cerenkov
effect is analogous to the sonic boom that occurs when
a macroscopic object such as a jet plane or a whip exceeds
the speed of sound in air. As the charged particle travels
through the medium, it loses kinetic energy by polarizing the
electrons of the insulator (typically water). These polarized
molecules then relax back to equilibrium through the
emission of ultraviolet and visible light, and when the speed
of the charged particle exceeds c, constructive interference
occurs, giving the observed Cerenkov radiation (6,7).
Although the use of Cerenkov radiation for scintillation
counting has been described (8–11), the use of inherent
light emission of radionuclides for in vivo imaging is a new
concept (12). In a recent paper, Robertson et al. were the
first to characterize the use of Cerenkov radiation for the
optical imaging of18F-labeled radiotracers in vivo (13).
Further work by Cho et al. (14) and Spinelli et al. (15)
verified the origins of visible light emission and paved the
way for the development of Cerenkov luminescence imag-
ing (CLI) as a novel in vivo imaging tool.
In this work, we provide further validation of the use of
Cerenkov radiation from a much larger range of radionu-
clides including the positron emitters18F,64Cu,89Zr, and
124I; b-emitter131I; and a-particle emitter225Ac. We report
in vitro phantom studies that compare the relative intensity
of the optical emission observed from these radionuclides
and demonstrate the linear correlation between the ob-
served light output and the measured PET signal. In
addition, we also report the feasibility of using CLI for
both the qualitative and the quantitative assessment of
radiopharmaceutical uptake in tumors in vivo. Uptake of
the novel monoclonal antibody (mAb)–based radiopharma-
ceutical89Zr-desferrioxamine B [DFO]-J591 for in vivo
immunoimaging of prostate-specific membrane antigen
(PSMA) expression in a clinically relevant model of
prostate cancer was observed by standard immuno-PET
and acute biodistribution studies. The results of these
studies are correlated with the tumor uptake observed by
CLI. These investigations reveal that optical imaging of
Cerenkov radiation shows excellent promise as a potential
new in vivo imaging modality for the rapid, low-cost, high-
throughput screening of radiopharmaceuticals.
MATERIALS AND METHODS
Full details of all methods and equipment used are presented in
the supplemental materials (available online only at http://
The radionuclides18F,89Zr, and124I were produced in high
radiochemical and radionuclidic purity via the18O-H2O(p,n)18F,
89Y(p,n)89Zr, and124TeO2(p,n)124I transmutation reactions on an
Ebco TR19/9 variable beam–energy cyclotron (Ebco Industries
Inc.) in accordance with previously reported methods (16–20).
64Cu was supplied by the Washington University School of
Medicine (21).131I was purchased as a131I-NaI (aqueous) solution
from MDS Nordion.225Ac was provided as a generous gift from
Dr. Michael R. McDevitt and was obtained as a carrier-free
product from elution of a229Th generator system (Oak Ridge
National Laboratory) (22). For mAb radiolabeling studies, the
89Zr-oxalate reagent was isolated in high radionuclidic and
radiochemical purity greater than 99.9%, with an effective specific
activity of 195–497 MBq/mg (5.28–13.43 mCi/mg) (18).
For each radionuclide studied, phantoms of varying activity
concentration (activity of 0–8.14 MBq [0–220 mCi] in 200 mL of
water, for a concentration of 0–40.7 kBq/mL [0–1.1 mCi/mL]) and
composed of 6 transparent plastic Eppendorf tubes were prepared
by 5 serial 1:2 dilutions of a known amount of activity in
deionized water. Phantoms were imaged at various times appro-
priate to the half-life (t1/2) of the radionuclide under investigation
using the Xenogen Ivis 200 device (Caliper Life Sciences). Where
possible, PET images of the phantoms were also recorded at the
same time points using a microPET Focus 120 scanner (Concorde
Antibody Conjugation and89Zr Radiolabeling
The IgG1mAb J591 was conjugated to the tris-hydroxamate,
hexadentate chelate DFO (Calbiochem) using a 6-step procedure
modified (24) from that described by Verel et al. (19) (supple-
All animal experiments were conducted in compliance with
Institutional Animal Care and Use Committee guidelines and the
Guide for the Care and Use of Laboratory Animals (25). All
animal procedures were performed under anesthesia by inhalation
of a 1%22% isoflurane (Baxter Healthcare)–oxygen mixture. Full
details are presented in the supplemental materials.
PET experiments were conducted on a microPET Focus 120
scanner (23). PET images of the in vitro phantoms were recorded
using the same methods and instrument parameters as described
for the small-animal immuno-PET studies.
Mice were administered89Zr-DFO-J591 formulations (10.9–
11.3 MBq [295–305 mCi], 60–62 mg of mAb, in 200 mL of sterile
saline for injection) via retroorbital injection. Approximately
1124THE JOURNAL OF NUCLEAR MEDICINE • Vol. 51 • No. 7 • July 2010
5 min before PET images were recorded, mice were anesthetized
by inhalation of a 1%22% isoflurane–oxygen gas mixture and
placed on the scanner bed. PET images were recorded at various
times between 24 and 96 h after injection. List-mode data were
acquired for between 10 and 30 min using a g-ray energy window
of 350–750 keV and a coincidence timing window of 6 ns (the
supplemental materials provide additional details).
Optical images were acquired using the Xenogen Ivis 200
optical imager. Cerenkov radiation was detected from each
phantom containing various activities of the same radionuclide
using the bioluminescence setting (integration time, 10, 20, 30, 40,
50, and 60 s; f/stop,1; binning, medium; field of view, B), with no
light interference from the excitation lamp. Spectral analysis was
obtained by measuring optical images either with or without the
use of a narrow band filter (560, 580, 600, 620, 640, and 680, or
open filter) of 20 nm in full width at half maximum (the
supplemental materials provide additional details).
Acute Biodistribution Studies
Acute in vivo biodistribution studies were conducted at the end
of the optical imaging and immuno-PET to validate the uptake and
localization of89Zr-DFO-J591 observed in mice bearing dual
subcutaneous LNCaP (50–250 mm3) tumors (n 5 3). Full details
are presented in the supplemental materials.
Data were analyzed using the unpaired, 2-tailed Student t test.
Differences at the 95% confidence level (P , 0.05) were con-
sidered to be statistically significant.
The ability to visualize and quantify the Cerenkov
radiation emitted from a range of positron, b-, and
a-emitting radionuclides was first investigated using in
vitro phantom studies. Standard solutions of decreasing
activity concentration (ranging from 0 to 40.7 kBq/mL) of
each radionuclide in water (200 mL) were prepared by 1:2
serial dilution and subjected to optical imaging at various
times appropriate for the t1/2 of the nuclide under in-
vestigation. For the positron-emitting radionuclides, the
phantoms were also imaged using PET. Typical optical and
PET images of the phantom are shown in Figures 1A and
1B, respectively. The phantom images recorded with the
other radionuclides18F,64Cu,89Zr,124I,131I, and225Ac
were qualitatively equivalent. However, for reasons of
clarity and consistency with the in vivo studies, our analysis
and discussion focus on89Zr.
The data acquired from the optical and PET phantom
studies were also quantitatively analyzed. Figure 2A shows
a plot of the average (background-corrected) radiance (p/s/
cm2/sr) versus the89Zr activity concentration (kBq/mL).
Background correction was applied by subtracting the
radiance measured from a region of interest (ROI) of the
optical images drawn over the sample containing no
activity. Linear regression analysis reveals a strong, positive
correlation between the light emission intensity and activity
concentration, with a correlation coefficient of R 5 0.98.
Figure 2B displays a plot of the normalized radiance versus
time, t/h, for the first two89Zr samples (Fig. 1, tubes 1 and
2). Exponential fitting of the data using the standard
equation for first-order radioactive decay gave an excellent
correlation (R 5 0.98), with a calculated t1/2(89Zr radiance)
equal to 79.1 6 4.8 h. This experimentally measured t1/2is
consistent with the known rate of decay of89Zr (t1/25
78.41 h), which provides additional evidence that the
source of the radiation arises directly from the radionuclide
decay (16). Furthermore, the optical emission profile
observed for18F,64Cu,89Zr,124I,131I, and225Ac was
found to be the same as previously reported for18F and is
consistent with Cerenkov radiation (13–15).
To assess the potential for using the observed optical
emission of radionuclides for quantitative analysis of the
images, the relationship between optical ROI and PET
volume of interest was examined. Figure 2C shows a plot of
the average radiance (p/s/cm2/sr) versus the mean activity
measured by PET (presented in units of percentage injected
dose per gram [%ID/g], which are commonly used for in
vivo analysis of radiotracer uptake). A linear relationship
(R 5 0.98) was observed between the optical and PET
signal intensities, suggesting that CLI is, in principle,
quantitative. The relationship presented here represents an
in vitro system for which the effects of depth- and medium-
dependent scattering are expected to be minimized. For in
vivo imaging, tissue penetration and scattering of light in
the ultraviolet and visible regions of the spectrum will
complicate the quantification of Cerenkov emission data.
To assess the relative utility of different radionuclides for
use in CLI, we investigated the relative light output from
each of the available radionuclides. Figure 3 shows a plot of
the ratio of the background-corrected average radiance to
the activity concentration (in units of [p/s/cm2/sr]/[kBq/
mL]) versus the radionuclide. To facilitate comparison, the
positron-emitting radionuclides have been arranged in order
of increasing mean b1kinetic energy/keV. The relationship
between the number of positrons emitted in a given energy
range and the theoretic number of photons produced in
imaging (A) and PET (B) of 6 samples of89Zr activity in water.
At time 0 h, the Eppendorf tubes labeled 1–6 corresponded
to activity concentrations of 40.3, 32.6, 27.4, 20.4, 13.3, and
0.00 kBq/mL. Optical images were recorded using integra-
tion time of 30 s and f/stop of 1.
Phantom images recorded using optical (CLI)
CERENKOV IMAGING OF MEDICAL ISOTOPES • Ruggiero et al. 1125
a medium of known refractive index is well established
(6,15,26). Despite the fact that the threshold for producing
coherent Cerenkov radiation in water is 263 keV (6,7),18F
with a mean b1kinetic energy of 249.8 keV and positron
yield of Ib
output comparable to the higher-energy emitter89Zr (Eb
395.5 keV; Ib
15 100% gives a measured radiance for light
15 22.7%). As expected for the positron-
emitting radionuclides, the higher-energy decay of124I was
found to give the most intense Cerenkov radiation.
225Ac was found to give the most intense optical
radiation (Fig. 3).225Ac is a pure (100%) a-emitter, and
its decay releases a-particles with energies in the range of
5,021 to 5,830 keV. This energy range is considerably
higher than the energy of the most energetic positrons
studied in this work (124I: Eb
However, because of their relatively large size (mass),
a-particles are known to travel at velocities below the
threshold for Cerenkov radiation (27). The origins of the
optical emissions observed from225Ac remain uncertain,
although it is possible that the optical emissions originate
from a series of short-lived, b-emitting daughter nuclides
including213Bi (t1/25 45.59 min, Eb
656 keV, Ib
The decay of
225Ac cannot be measured by current
nuclear imaging modalities. Validation of the use of
225Ac for optical imaging provides an example of the
potential applications of dedicated CLI devices. A range
of225Ac-labeled mAb-based agents has been developed
(28–31). In an ongoing phase I clinical trial between
Memorial Sloan-Kettering Cancer Center (MSKCC) and
the National Cancer Institute,
humanized anti-CD33 mAb225Ac-HuM195 is under eval-
uation as a radioimmunotherapeutic agent for targeted
therapy of leukemia and myelodysplastic syndrome (clinical
trial NCT00672165). The ability to measure the tumor
targeting of225Ac-labeled mAbs and potentially estimate
1[maximum] 5 2,137.6 keV).
2[mean] 5 435 keV,
2[mean] 5 197.5
25 97.8%),209Pb (t1/25 3.253 h, Eb
25 100%), and209Tl (t1/25 2.20 min, Eb
25 100%) (28).
225Ac conjugated to the
Positive correlation observed between measured average
radiance (background-corrected in units of p/s/cm2/sr) and
89Zr activity concentration (kBq/mL) (A), rate of decay
observed in normalized radiance vs. time/h (B), and linear
correlation observed between average radiance (back-
ground and decay-corrected in units of p/s/cm2/sr) vs.
mean PET signal intensity (measured in units of %ID/g,
commonly used for quantification of in vivo PET studies) (C).
Ave. 5 average; Exp. 5 exponential.
Quantitative analysis of phantom studies.
activity concentration (mCi/mL) vs. radionuclide. Positron-
emitting radionuclides are arranged in order of increasing
average b1kinetic energy (18F: Eb
395.5 keV [Ib
a-particle emission with Eain range of 5,021–5,830 keV (16).
Ave. 5 average.
Plot of ratio of average radiance (p/s/cm2/sr)/
15 249.8 keV [Ib
15 17.6%];89Zr: Eb
15 820 keV [Ib
2100%).225Ac decays by 100%
15 278.2 keV [Ib
15 22.7%];124I: Eb
25 181.9 keV (Ib
1126THE JOURNAL OF NUCLEAR MEDICINE • Vol. 51 • No. 7 • July 2010
in vivo dosimetry using optical imaging would represent
a fundamental advance in imaging science.
To assess the potential of optical CLI of tumors in vivo,
we developed the89Zr-labeled mAb89Zr-DFO-J591 (32–
36). Several examples of the radiolabeling, characteriza-
tion, and use of
89Zr-DFO-mAbs for immuno-PET of
various cancers have been reported (24,37–41).
In these studies, the humanized mAb J591, which binds
to an extracellular epitope of PSMA expressed in most
prostate cancer cell lines, was functionalized with the tris-
hydroxamate chelate DFO using bioconjugation methods
modified (24) from the pioneering work of Verel et al. (19).
Full details of the mAb conjugation, identification, and89Zr
radiolabeling and in vitro and in vivo characterization of
89Zr-DFO-J591 will be reported elsewhere. The final
radiochemical yield of the purified
67%, and the product was formulated in 0.9% sterile saline
with a radiochemical purity greater than 99% and a specific
activity of 165.0 MBq/mg (4.47 mCi/mg) of mAb (Sup-
plemental Figs. 1 and 2).
In Vivo Studies
The site-specific localization of89Zr-DFO-J591 in sub-
cutaneous human xenograft LNCaP (PSMA-positive) tu-
mors was used to assess the ability of optical CLI and
immuno-PET to provide both qualitative and quantitative
data on the biodistribution of a radiotracer in vivo.
Temporal images of
89Zr-DFO-J591 (10.9–11.3 MBq
[295–305 mCi], 60–62 mg of mAb in 200 mL of sterile
saline) tumor uptake recorded between 24 and 96 h after
retroorbital administration using CLI and immuno-PET are
presented in Figures 4A and 4B, respectively. The mice
were shaved to reduce signal scattering before imaging at
24 h (15). The images in Figure 4A demonstrate that optical
imaging of Cerenkov radiation derived from the decay of
an administered radiotracer can be achieved in vivo. In
these studies each mouse was inoculated on both the right
and the left flanks with LNCaP cells. Tumors grew on both
flanks, but in each animal the growth of 1 tumor exceeded
that of the other by between 3- and 4-fold, providing 2
different datasets classified as either small or large LNCaP
tumors. Figure 4A also shows that differential uptake
between the small and large tumors can be discerned by
sterile saline) recorded in dual subcutaneous LNCaP (PSMA-positive) tumor–bearing severe combined immune deficient mice
between 24 and 96 h after administration. (A) Signal observed in optical spectrum from in vivo CLI of89Zr-DFO-J591 tumor
uptake in 3 mice. (B) Corresponding coronal and transverse immuno-PET images recorded for mouse 3. (C) Optical image
recorded of organs after acute ex vivo biodistribution at 96 h. Transverse and coronal planar immuno-PET images intersect
center of tumors. Upper and lower thresholds of CLI and immuno-PET images in A–C have been adjusted for visual clarity, as
indicated by scale bars. Trans. 5 transverse; 1ve 5 positive; T(L) 5 left tumor; T(R) 5 right tumor; He 5 heart; Lu 5 lungs; Li 5
liver; Sp 5 spleen; Ki 5 kidneys; L. Int. 5 large intestine; Bo 5 bone; Mu 5 muscle.
Temporal images of89Zr-DFO-J591 uptake (10.9–11.3 MBq [295–305 mCi], 60–62 mg of mAb, in 200 mL of 0.9%
CERENKOV IMAGING OF MEDICAL ISOTOPES • Ruggiero et al.1127
optical CLI. This result is, to the best of our knowledge, the
first demonstration of specific tumor imaging using the
inherent Cerenkov radiation emitted from a metallolabeled
immunoconjugate. Furthermore, qualitative analysis of the
CLI pictures indicates that higher89Zr-DFO-J591 back-
ground activity is present at 24 h, with lower uptake in the
tumors. Between 24 and 96 h, tumor uptake of89Zr-DFO-
J591 continues to increase and activity in the background
shows a concordant decrease (as deduced by observation of
ROIs located over the mice but remote from the tumor
89Zr-DFO-J591 temporal immuno-
PET images of a representative animal (mouse 3) recorded
at the same times as the CLI pictures are shown in Figure
4B. Transverse and coronal slices are taken through the
center of the tumors. For mouse 3, the LNCaP tumor
located in the right flank was approximately 3 times larger
in volume (250 mm3) than the tumor in the left flank
(80 mm3). The first observation is that
provides excellent contrast for the delineation of tumor-
versus-background tissue uptake using immuno-PET. Fur-
thermore, facile distinction between the radiotracer uptake
and accumulation in the small and large tumors was
observed, and tumor uptake continued to increase during
the full time course of the immuno-PETexperiments. These
immuno-PET studies provide a reference point for the
interpretation of the CLI data and confirm that the observed
qualitative increase in optical intensity of the tumor ROIs
over time is due to an increase in radiotracer accumulation.
Full quantitative analysis is discussed in the next sections.
At the end of the imaging experiment (96 h), the mice
were sacrificed and subjected to both optical imaging of the
excised organs and acute ex vivo biodistribution studies to
quantify the accumulation of89Zr radioactivity. An optical
image of the excised organs from mice 1–3 is shown in
Figure 4C. The organ image demonstrates that at the same
optical emission settings and threshold values as used for
the in vivo images, uptake in only the LNCaP tumors was
observed. The lack of89Zr activity in the optical image of
the background organs is consistent with the immuno-PET
images recorded at 96 h, suggesting excellent clearance of
89Zr-DFO-J591 from nontarget tissue.
Results from the acute biodistribution study at 96 h are
presented as a bar chart in Figure 5. The data confirm that
high uptake of89Zr-DFO-J591 in PSMA-positive tumors
occurs within 96 h after administration. Differential radio-
tracer uptake and accumulation were observed between the
large and small tumor groups. For the larger LNCaP tumors
(average volume, 220 mm3), radiotracer uptake reached
72.3 6 4.6 %ID/g. However, for the smaller tumors
(average volume, 65 mm3), radiotracer uptake was still
well above background tissue uptake but reached only 32.0 6
5.4 %ID/g (P 5 0.0007). The dependence of radiotracer
accumulation on tumor size is likely due to enhanced
vascularization and the increased number of available
PSMA epitopes presented by the well-established tumors.
Overall, these biodistribution data are consistent with the
optical and immuno-PET studies.
For an imaging modality to be useful in a clinical setting,
it is essential that the data obtained be at least semi-
quantitative. Figure 6 shows the time–activity curves de-
rived from ROI and volume-of-interest analysis of the in
vivo optical and immuno-PET images. The time–activity
curves confirm that89Zr-DFO-J591 is efficiently removed
from the blood pool and accumulated only to low levels in
background tissue. The time–activity curves also show that
89Zr-DFO-J591 uptake in the larger tumors increased
between 24 and 96 h. For example, the immuno-PET tumor
time–activity curve shows an increase in radiotracer accu-
mulation from 16.7 6 1.4 %ID/g at 24 h to 45.1 6 10.3 %
ID/g at 96 h, corresponding to a 2.5- 6 0.8-fold increase. ROI
tion data (%ID/g) for uptake of89Zr-DFO-J591 in male severe
combined immune deficient mice at end of optical and
immuno-PET experiments (96 h after injection). T 5 tumor.
Bar chart showing selected tissue biodistribu-
of-interest analysis of CLI and immuno-PET images for89Zr-
DFO-J591 uptake in well-established (large) LNCaP tumors.
Volume-of-interest analysis of immuno-PET images shows
89Zr activity in heart–blood pool and muscle
Time–activity curves showing ROI and volume-
1128THE JOURNAL OF NUCLEAR MEDICINE • Vol. 51 • No. 7 • July 2010
analysis of the optical images also showed the same increase
in radiotracer uptake in the larger tumors over time. The
measured, decay-corrected, average radiance for the larger
corresponds to a 1.8- 6 0.3-fold increase over time, which is
similar to changes observed from the immuno-PET studies.
Indeed, accounting for the relative errors in the 2 measure-
ments, the observed change in CLI intensity for89Zr-DFO-
J591 tumor accumulation was the same as for immuno-PET.
In 2009, Robertson et al. reported proof-of-concept stud-
ies that validated the use of Cerenkov radiation emissions
in the ultraviolet and visible regions of the spectrum for in
vivo optical imaging of18F-radiolabeled compounds (13).
This new technique was termed CLI and represents a
fundamental advance toward the development of hybrid
nuclear–optical tomographic imaging devices. In this work,
we explored the potential of imaging Cerenkov radiation
with a wider range of radionuclides and provided the first
examples of dual optical CLI and PET of in vivo tumor
uptake using a metallolabeled antibody.
The in vitro and in vivo imaging studies presented in this
work demonstrate not only that the inherent Cerenkov
emissions of various clinically relevant radionuclides can
be visualized but also that the data obtained correlate with
the observed biodistribution of radiotracers. In addition,
both qualitative and quantitative interpretation of the CLI
data was found to give a strong correlation with immuno-
PET and biodistribution studies. We anticipate that CLI of
administered radiopharmaceuticals has a broad range of
potential applications. In particular, the ability to simulta-
neously measure time-dependent changes in tumor uptake
of radiotracers in multiple different tumor models or che-
motherapeutic treatment regimes means that optical CLI
offers the potential to conduct rapid, low-cost, high-
throughput screening of novel radiotracers in vivo. In
addition, intraoperative imaging of Cerenkov radiation is
feasible with a highly sensitive camera in a dark room, the
use of which can be achieved much more easily than with
expensive nuclear medicine equipment. Notably, no addi-
tional development of optical agents is required because
CLI can take advantage of many approved radiopharma-
ceuticals. Therefore, the combination of endoscopic surgical
methods with optical Cerenkov imaging has the potential to
be used directly in the clinic for intraoperative visualization
of tumor lesions and margins or metastatic involvement for
Cerenkov radiation-guided surgery. Further studies using
hybrid nuclear–optical imaging of radiopharmaceuticals are
under way at MSKCC.
Basic characterization of the inherent Cerenkov radiation
emission from a range of positron-, b-, and a-emitting
radionuclides commonly used as imaging and therapeutic
isotopes in nuclear medicine is reported. Phantom studies
confirm that the broad-spectrum optical emissions arise
because of Cerenkov radiation in the condensed phase.
The measured radiance was found to give a strong correla-
tion with the known activity concentration, and the signal
intensity was found to correlate with the known t1/2of the
nuclides under investigation. In addition, in vitro studies
comparing the optical signal intensity with that observed
from the PET images of the positron-emitting nuclides
revealed a linear correlation, indicating that CLI has the
potential to provide quantitative data analysis of radiotracers.
In vivo studies looking at the uptake of89Zr-DFO-J591
in PSMA-positive LNCaP prostate tumors demonstrated
that for the first time, to our knowledge, Cerenkov radiation
emission can be used to quantify the tumor-specific uptake
of a novel targeted, metallolabeled tracer. Furthermore,
time–activity curves revealed that the intensity of the
observed optical signal correlated with the quantitative
immuno-PET and acute biodistribution studies. These re-
sults pave the way for further use and development of CLI
as a novel optical imaging modality for the rapid, cost-
effective, high-throughput screening of radiopharmaceuti-
cals. Studies aimed at using the Cerenkov radiation emitted
by various radionuclides in the development of activatable
reporter system probes are under way at MSKCC.
We thank Drs. NagaVaraKishore Pillarsetty, and Pat
Zanzonico for informative discussions; Valerie M. Longo
Vadim Divilov for advice on in vitro experiments; and
Bradley Beattie for assistance with the optical imaging. We
thank Dr. Neil H. Bander (Weill Medical College of Cornell
University) for the generous gift of J591. We also thank the
CA046945). Technical services provided by the MSKCC
Small-Animal Imaging Core Facility were supported in part
by NIH grants R24 CA83084 and P30 CA08748.
1. Weissleder R, Pittet MJ. Imaging in the era of molecular oncology. Nature. 2008;
2. Pichler BJ, Kolb A, Nagele T, Schlemmer H-P. PET/MRI: paving the way for the
next generation of clinical multimodality imaging applications. J Nucl Med.
3. Kirsch DG, Dinulescu DM, Miller JB, et al. A spatially and temporally restricted
mouse model of soft tissue sarcoma. Nat Med. 2007;13:992–997.
4. Grimm J, Kirsch DG, Windsor SD, et al. Use of gene expression profiling to
direct in vivo molecular imaging of lung cancer. Proc Natl Acad Sci. 2005;102:
5. Cerenkov PA. Visible emission of clean liquids by action of g-radiation. C R
Dokl Akad Nauk SSSR. 1934;2:451–454.
CERENKOV IMAGING OF MEDICAL ISOTOPES • Ruggiero et al.1129
6. Jelley JV. Cerenkov radiation and its applications. Br J Appl Phys. 1955;6: Download full-text
7. Ross HH. Measurement of b-emitting nuclides using Cerenkov radiation. Anal
8. Plesums J, Bunch WH. Measurement of phosphorus following32P Cerenkov
counting. Anal Biochem. 1971;42:360–362.
9. Simonnet F, Combe J, Simonnet G. Detection of32P scintillating plastic vials.
Appl Radiat Isot. 1987;38:311–312.
10. Hansen BS. Improved method for assaying pyrophosphate exchange measuring
Cerenkov radiation. Anal Biochem. 1980;109:12–17.
11. Berger SL. The use of Cerenkov radiation for monitoring reactions performed in
minute volumes: examples from recombinant DNA technology. Anal Biochem.
12. Miyata M, Tomita H, Watanabe K, Kawarabayshi J, Iguchi T. Development of
TOF-PET using Cherenkov radiation. J Nucl Sci Technol. 2006;43:339–343.
13. Robertson R, Germanos MS, Li C, Mitchell GS, Cherry SR, Silva MD. Optical
imaging of Cerenkov light generation from positron-emitting radiotracers. Phys
Med Biol. 2009;54:N355–N365.
14. Cho JS, Taschereau R, Olma S, et al. Cerenkov radiation imaging as a method for
quantitative measurements of beta particles in a microfluidic chip. Phys Med
15. Spinelli AE, D’Ambrosio D, Calderan L, Marengo M, Sbarbati A, Boschi F.
Cerenkov radiation allows in vivo optical imaging of positron emitting
radiotracers. Phys Med Biol. 2010;55:483–495.
16. Holland JP, Williamson MJ, Lewis JS. Unconventional nuclides for radiophar-
maceuticals. Mol Imaging. 2010;9:1–20.
17. Welch MJ, Redvanly CS, Editors. Handbook of Radiopharmaceuticals:
Radiochemistry and Applications. New York, NY: Wiley; 2003.
18. Holland JP, Sheh Y, Lewis JS. Standardized methods for the production of high
specific-activity zirconium-89. Nucl Med Biol. 2009;36:729–739.
19. Verel I, Visser GWM, Boellaard R, Stigter-van Walsum M, Snow GB, van
89Zr immuno-PET: comprehensive procedures for the
89Zr-labeled monoclonal antibodies. J Nucl Med. 2003;44:
20. Sheh Y, Koziorowski J, Balatoni J, Lom C, Dahl JR, Finn RD. Low energy
cyclotron production and chemical separation of ‘‘no carrier added’’ iodine-124
from a reusable, enriched tellurium-124 dioxide/aluminum oxide solid solution
target. Radiochim Acta. 2000;88:169–173.
21. McCarthy DW, Shefer RE, Klinkowstein RE, et al. Efficient production of high
specific activity64Cu using a biomedical cyclotron. Nucl Med Biol. 1997;24:35–
22. Miederer M, Scheinberg DA, McDevitt MR. Realizing the potential of the
actinium-225 radionuclide generator in targeted alpha particle therapy
applications. Adv Drug Deliv Rev. 2008;60:1371–1382.
23. Kim JS, Lee JS, Im KC, et al. Performance measurement of the microPET Focus
120 scanner. J Nucl Med. 2007;48:1527–1535.
24. Holland JP, Caldas-Lopes E, Divilov V, et al. Measuring the pharmacokinetic
effects of a novel Hsp90 inhibitor on HER2/neu expression in mice using89Zr-
DFO-trastuzumab. PLoS One. 2010;5:e8859.
25. Guide for the Care and Use of Laboratory Animals. Washington, DC: National
Academy Press; 1996.
26. Levin CS, Hoffman EJ. Calculation of positron range and its effects on the
fundamental limit of positron emission tomography system spatial resolution.
Phys Med Biol. 1999;44:781–799.
27. Cherry SR, Sorenson JA, Phelps ME. Physics in Nuclear Medicine. 3rd ed.
Philadelphia, PA: Saunders; 2003.
28. McDevitt MR, Scheinberg DA. Ac-225 and her daughters: the many faces of
Shiva. Cell Death Differ. 2002;9:593–594.
29. McDevitt MR, Ma D, Lai LT, et al. Tumor therapy with targeted atomic
nanogenerators. Science. 2001;294:1537–1540.
30. Singh Jaggi J, Henke E, Seshan SV, et al. Selective alpha-particle mediated
depletion of tumor vasculature with vascular normalization. PLoS One. 2007;2:
31. Ballangrud A˚M, Yang W-H, Palm S, et al. Alpha-particle emitting atomic
generator (actinium-225)-labeled trastuzumab (Herceptin) targeting of breast
cancer spheroids: efficacy versus HER2/neu expression. Clin Cancer Res. 2004;
32. Liu H, Moy P, Kim S, et al. Monoclonal antibodies to the extracellular domain of
prostate-specific membrane antigen also react with tumor endothelium. Cancer
33. Liu H, Rajasekaran AK, Moy P, et al. Constitutive and antibody-induced
internalization of prostate-specific membrane antigen. Cancer Res. 1998;58:
34. Smith-Jones PM, Vallabahajosula S, Goldsmith SJ, et al. In vitro characterization
of radiolabeled monoclonal antibodies specific for the extracellular domain of
prostate-specific membrane antigen. Cancer Res. 2000;60:5237–5243.
35. Smith-Jones PM, Vallabhajosula S, Navarro V, Bastidas D, Goldsmith SJ, Bander
NH. Radiolabeled monoclonal antibodies specific to the extracellular domain of
prostate-specific membrane antigen: preclinical studies in nude mice bearing
LNCaP human prostate tumor. J Nucl Med. 2003;44:610–617.
36. McDevitt MR, Barendswaard E, Ma D, et al. An a-particle emitting antibody
([213Bi]J591) for radioimmunotherapy of prostate cancer. Cancer Res. 2000;60:
37. Verel I, Visser GWM, Boellaard R, et al. Quantitative89Zr immuno-PET for in
vivo scouting of90Y-labeled monoclonal antibodies in xenograft-bearing nude
mice. J Nucl Med. 2003;44:1663–1670.
38. Perk LR, Visser OJ, Stigter-van Walsum M, et al. Preparation and evaluation of
89Zr-Zevalin for monitoring of
90Y-Zevalin biodistribution with positron
emission tomography. Eur J Nucl Med Mol Imaging. 2006;33:1337–1345.
39. Borjesson PKE, Jauw YWS, de Bree R, et al. Radiation dosimetry of89Zr-
labeled chimeric monoclonal antibody U36 as used for immuno-PET in head and
neck cancer patients. J Nucl Med. 2009;50:1828–1836.
40. Aerts HJWL, Dubois L, Perk L, et al. Disparity between in vivo EGFR
expression and89Zr-labeled cetuximab uptake assessed with PET. J Nucl Med.
41. Dijkers ECF, Kosterink JGW, Rademaker AP, et al. Development and
characterization of clinical-grade89Zr-trastuzumab for HER2/neu immunoPET
imaging. J Nucl Med. 2009;50:974–981.
1130THE JOURNAL OF NUCLEAR MEDICINE • Vol. 51 • No. 7 • July 2010