What Is Molecular Imaging?

Molecular imaging is a type of medical imaging that provides detailed pictures of what is
happening inside the body at the molecular and cellular level. Where other diagnostic imaging
proceduressuch as x-rays, computed tomography (CT) and ultrasoundpredominantly offer
anatomical pictures, molecular imaging allows physicians to see how the body is functioning and
to measure its chemical and biological processes.
Molecular imaging techniques depend upon molecular mechanisms operative in vivo. This
imaging technique encompasses the visualization, characterization and measurement of
biological processes at the molecular and cellular levels in humans and other living systems. The
techniques used include Positron Emission Tomography Computed Tomography (PET-CT),
nuclear medicine, Magnetic Resonance Imaging (MRI), Magnetic Resonance Spectroscopy
(MRS), optical imaging and ultrasound. There are escalating evidences in the published data that
discussed the advantages of integrated molecular imaging technique as an accurate tool in
localizing abnormal metabolic alteration and serve as a potential role as an invasive surrogate
biomarker for various disease entities.
It also plays an increasingly fundamental role in drug discovery and early development in
humans. The evolution of molecular imaging tool in specific PET-CT has impacted the use of
molecular imaging technique in many altered cellular mechanism. In particular, PET which was
introduced in the 1970 s is capable of quantifying individual changes on the different pathology
that underpin the biological reprogramming in abnormal cells. Intensive research activities in
various PET applications gradually evolved to its clinical use first in neuropsychiatric disorders
and cardiology, then in oncology. Molecular imaging provides the key to the future of
personalized medicine, which involves diagnosing, treating and monitoring patients based on
their individual makeup. The amelioration in its technique has braced the one-stop-imaging
strategy in various disease entities as a tool for disease localization, prediction and treatment
monitoring. For the purpose of the discussion in this chapter, we highlight the integrated
molecular imaging technique PET-CT employing flurodeoxyglucose (FDG) as a standard of care
utility in various disease pathology.
Molecular imaging offers unique insights into the human body that enable physicians to
personalize patient care. In terms of diagnosis, molecular imaging is able to:

Provide information that is unattainable with other imaging technologies or that would
require more invasive procedures such as biopsy or surgery.
Identify disease in its earliest stages and determine the exact location of a tumor, often
before symptoms occur or abnormalities can be detected with other diagnostic tests.

As a tool for evaluating and managing the care of patients, molecular imaging studies help
physicians:

Determine the extent or severity of the disease, including whether it has spread elsewhere
in the body.

Select the most effective therapy based on the unique biologic characteristics of the
patient and the molecular properties of a tumor or other disease.
Determine a patients response to specific drugs.
Accurately assess the effectiveness of a treatment regimen.
Adapt treatment plans quickly in response to changes in cellular activity.
Assess disease progression.
Identify recurrence of disease and help manage ongoing care.

Molecular imaging procedures, which are noninvasive, safe and painless, are used to diagnose
and manage the treatment of:

Cancer
Heart disease
Brain disorders, such as Alzheimers disease
Gastrointestinal disorders
Lung disorders
Bone disorders
Kidney and thyroid disorders

How does molecular imaging work?

When disease occurs, the biochemical activity of cells begins to change. For example, cancer
cells multiply at a much faster rate and are more active than normal cells. Brain cells affected by
dementia consume less energy than normal brain cells. Heart cells deprived of adequate blood
flow begin to die.
As disease progresses, this abnormal cellular activity begins to affect body tissue and structures,
causing anatomical changes that may be seen on CT or magnetic resonance (MR) scans. For
example, cancer cells may form a mass or tumor. With the loss of brain cells, overall brain
volume may decrease or affected parts of the brain may appear different in density than the
normal areas. Similarly, the heart muscle cells that are affected stop contracting and the overall
heart function deteriorates.
Molecular imaging excels at detecting the cellular changes that occur early in the course of
disease, often well before structural changes can be seen on CT and MR images.
Most molecular imaging procedures involve an imaging device and an imaging agent, or probe.
A variety of imaging agents are used to visualize cellular activity, such as the chemical processes
involved in metabolism, oxygen use or blood flow. In nuclear medicine, which is a branch of
molecular imaging, the imaging agent is a radiotracer, a compound that includes a radioactive
atom, or isotope. Other molecular imaging modalities, such as optical imaging and molecular
ultrasound, use a variety of different agents. Magnetic resonance spectroscopy is able to measure
chemical levels in the body, without the use of an imaging agent.
Once the imaging agent is introduced into the body, it accumulates in a target organ or attaches
to specific cells. The imaging device detects the imaging agent and creates pictures that show
how it is distributed in the body. This distribution pattern helps physicians discern how well
organs and tissues are functioning.

How is molecular imaging performed?

Molecular imaging procedures are often performed on an outpatient basis. If an imaging agent is
needed, it is injected, swallowed or inhaled. Imaging is performed, depending on the procedure,
immediately or hours or even days afterward, depending on the type of procedure. Images
created by the device and a computer are reviewed and interpreted by a qualified imaging
professional, such as a nuclear medicine physician or radiologist, who shares the results with the
patients physician.

What molecular imaging technologies are used today?

Gamma camera
A gamma camera is a specialized camera that is capable of detecting a radiotracer. The gamma
camera creates Two-dimensional pictures of the inside of the body from different angles.
Nuclear imaging or also called as radionuclide scanning provides an effective diagnostic tools
for the radiologists as it shows not only the structure of an organ but also the function of the
organ. Nuclear imaging routine uses small amounts of radioactive material, or tracer for
diagnostic purpose. Radioactive tracer used in nuclear imaging is normally a specifically targeted
probe. It could be antibodies, ligands or substrates to specifically interact with protein targets in
particular cells or sub cellular compartments. These interactions are based on either receptorradioligand binding or enzyme mediated trapping of a radio labeled substrate. Radioactive
tracers used in nuclear imaging are in most cases is administered into a vein and some are given
orally. After an administration of radioactive tracers, patient is required to rest for a certain
period to allow distribution of radioactive tracer in the body. In the end, for imaging purpose, a
specialized gamma camera is used to detect the radiation throughout the body. Most commonly
used techniques in nuclear imaging are positron emission tomography (PET) and single photon
emission computed tomography (SPECT).
Positron emission tomography (PET)
PET involves the use of an imaging device (PET scanner) and a radiotracer that is injected into
the patients bloodstream. A frequently used PET radiotracer is 18F-fluorodeoxyglucose (FDG),
a compound derived from a simple sugar and a small amount of radioactive fluorine.
Once the FDG radiotracer accumulates in the bodys tissues and organs, its natural decay
includes emission of tiny particles called positrons that react with electrons in the body. This
reaction, known as annihilation, produces energy in the form of a pair of photons. The PET
scanner, which is able to detect these photons, creates three-dimensional images that show how
the FDG is distributed in the area of the body being studied.
Areas where a large amount of FDG accumulates, called hot spots because they appear more
intense than surrounding tissue, indicate that a high level of chemical activity or metabolism
is occurring there. Areas of low metabolic activity appear less intense and are sometimes referred
to as cold spots. Using these images and the information they provide, physicians are able to
evaluate how well organs and tissues are working and to detect abnormalities.

PET-CT is a combination of PET and CT that produces highly detailed views of the body. The
combination of two imaging techniquescalled co-registration, fusion imaging or hybrid
imagingallows information from two different types of scans to be viewed in a single set of
images. CT imaging uses advanced x-ray equipment and in some cases a contrast-enhancing
material to produce three-dimensional images.
A combined PET-CT study is able to provide detail on both the anatomy and function of organs
and tissues. This is accomplished by superimposing the precise location of abnormal metabolic
activity (from PET) against the detailed anatomic image (from CT).
Single photon emission computed tomography (SPECT)
Similar to PET, single photon emission computed tomography (SPECT) also uses a radioactive
tracer that is administered to the patient and a scanner to record data that a computer constructs
into two or three dimensional images. However, in another note, SPECT technique employs a
gamma camera that rotates around the patient to detect a radioactive tracer in the body. In
contrast to PET which employs shorter half-lived tracers as opposed to the SPECT tracers? If a
tumor is present, the antibodies will stick to it and thus allow for detection of timorous cells. For
better understanding on strength and weakness of each imaging modalities.
A SPECT scan uses a gamma camera that rotates around the patient to detect a radiotracer in the
body. Working with a computer, SPECT creates three-dimensional images of the area being
studied. SPECT may also be combined with CT for greater accuracy.
Magnetic resonance spectroscopy
Magnetic resonance imaging or popularly known as MRI is an imaging technique used mainly in
medical settings to produce high quality images of inside human body. Theoretically, the
mechanism behind MRI is based on the principles of nuclear magnetic resonance (NMR), a
spectroscopic technique used by scientist to obtain microscopic chemical and physical
information about molecules. MRI scanner has a tube surrounded by a giant circular magnet.
During routine examination of MRI, patient is placed on a moveable bed that is inserted into the
magnet. The presence of magnet creates a strong magnetic field that aligns the protons of
hydrogen atoms, which are then exposed to a beam of radio waves. This spins various protons of
the body, and produces a faint signal that is detected by the receiver portion of the MRI scanner.
The receiver information is processed by a computer and an image is then produced.
MR imaging uses a magnetic field, radio waves and a computer to create detailed images of the
body. MR spectroscopy uses MR to measure metabolites, which are substances produced by
chemical reactions in the brain and other areas of the body. MR spectroscopy provides
information on the location of specific chemicals in the body and on biochemical activity within
cells.

Optical imaging
In optical imaging, light-producing proteins are designed to attach to specific moleculessuch
as brain chemicals or molecules on the surface of cancer cells. Highly sensitive detectors are able
to detect low levels of light emitted by these molecules from inside the body. The two major
types of optical imaging are:

Bioluminescent imaging, which uses a natural light-emitting protein (luciferase, for

example, the substance that produces the glow of fireflies) to trace the movement of
certain cells or to identify the location of specific chemical reactions within the body.
Fluorescence imaging, which uses proteins that produce light when activated by an
external light source such as a laser.

Molecular ultrasound imaging

Ultrasound imaging has been used for over 20 years. It uses high-frequency sound waves to view
soft tissues such as muscles and internal organs inside the body. As the image of the ultrasound
is captured in real-time, it enables the physician to see the movement of the bodys internal
organs as well as blood flowing through the vessels. In an ultrasound exam, a handheld
transducer is placed against the skin. The transducer sends out high frequency sound waves that
reflect off the body structures. As sound waves directed through the body bounce back when
they encounter different tissues, echoes are measured with the help of a computer and are
converted into real-time images of organs and tissues. The image captured is based on the
frequency and strength of the sound signal and the time it takes to return from the patient to the
transducer.
Traditional ultrasound imaging uses high-frequency sound waves to produce pictures of the
inside of the body. As sound waves directed through the body bounce back when they encounter
different tissues, echoes are measured with the help of a computer and are converted into realtime images of organs and tissues. Molecular ultrasound uses targeted micro bubbles, extremely
small, hollow structures that serve as a contrast agent during an ultrasound exam.

How safe is molecular imaging?

Molecular imaging procedures are noninvasive and very safe. When used, the amount of
radioactivity used in nuclear medicine procedures is very small. The radiation risk is very low
compared with the potential benefits. There are no known long-term side effects from nuclear
medicine procedures, which have been performed for more than 50 years. Allergic reactions may
occur but are extremely rare and usually mild. Imaging agents are approved by the U.S. Food
and Drug Administration.

What is the future of molecular imaging?

In addition to increasing our understanding of the underlying causes of disease, molecular
imaging is improving the way disease is detected and treated. Molecular imaging technologies
are also playing an important role in the development of:

Screening tools, by providing a non-invasive and highly accurate way to assess at-risk
populations.
New and more effective drugs, by helping researchers quickly understand and assess new
drug therapies.

Personalized medicine, in which medical treatment is based on a patients unique genetic

profile.

In the future, molecular imaging will include an increased use of:

Fusion or hybrid imaging, in which two imaging technologies are combined to produce
one image.
Optical imaging
New probes for imaging critical cancer processes.
Reporter-probe pairs that will facilitate molecular-genetic Imaging.

MOLECULAR IMAGING STRATEGIES

The imaging strategies most widely used can be classified as direct and indirect. Direct
molecular imaging is characterized by the direct and specific interaction of the molecular probe
with a target, resulting in probe localization, accumulation, and/or activation.
As shown in Figure 2, the target can be a cell surface receptor, antigen, enzyme, transporter,
channel, or nucleotide. Targets can be located on the cell surface or cell periphery, or in
organelles or the nucleus of the cell. Probes designed for direct imaging are highly specific and
can be used to interrogate only the intended molecular target. Hence, for each novel target, a new
probe must be developed, characterized, and validated, all of which are time-consuming and
costly endeavors. The second strategy embodies a more generalized and flexible approach in
which established imaging systems can be linked to various molecular processes of interest and
indirectly report on their activity.
As illustrated in Figure 3, reporter gene imaging, the most common practice of indirect imaging,
entails expression of a reporter gene encoding for a targetable receptor, enzyme, or transporter on
the occurrence of a specific molecular event. Complementary reporter probes are then infused
systemically andinstead of interacting with endogenous targets as in direct imagingare
bound, trapped, or activated by the exogenous reporter gene product to generate a detectable
signal. In some instances, the reporter gene product itself provides the imaging signal (eg,
fluorescent proteins), obviating reporter probes. Reporter gene products therefore act as
surrogate markers for the level of expression of the protein being studied. Although reporter gene
systems can be engineered to interrogate a variety of biologic events, they are constrained by the
need for genetic manipulation of the tissue being studied. In all cases, the reporter gene construct
must be first introduced into the cells of living subjects.
Direct Imaging
The direct strategy is used across all four imaging modalities and can be further categorized by
the specific target type (Fig 2). Careful target selection is critical for any direct imaging study to
be informative. Naturally, the target should play an important role in the initiation or progression
of the disease pathway being studied and should also be available in sufficiently high quantities
to ensure generation of a detectable signal. Imaging the end products of gene expression is
therefore preferred because transcription and translation are intrinsic amplification mechanisms.
Imaging cell surface receptors and antigens is a common form of direct imaging used in all
imaging modalities. Direct imaging of enzymes entails enzymemediated trapping of a labeled
substrate or activation of a silent probe. PET with FDG, a widely known example of the former,
specifically images the activity of hexokinase as an indicator of glucose use and metabolic
activity. FDG is transported into cells by glucose transporters and then phosphorylate by
hexokinase to FDG-6- phosphate, a modification that prevents its diffusion out of cells. Relative
to receptor imaging, direct enzyme imaging provides inherent signal amplification because
enzymes can act on multiple probes, whereas receptors generally interact with only a single
probe. By contrast, direct imaging of messenger RNA has been particularly challenging, given
the relatively low number of target messenger RNA available per cell.

Activatable Probes
The aforementioned approaches to direct imaging often suffer from high background noise.
Because these probes emit signal constitutively, it is not possible to differentiate probes that have

reached their target from those that have not. This necessitates a time delay between tracer
injection and imaging to allow washout of excess unbound probes from the tissue and systemic
circulation. Despite this, significant background noise can remain as a result of nonspecific
binding. To circumvent this problem, activatable probes (also called smart probes or
molecular beacons) have been developed for optical and MR imaging. Such agents are
designed to emit signal only after activation by the intended target, usually an enzyme. It is
estimated that this strategy, through background suppression, amplifies signal generation as
much as several hundred fold.
As illustrated in Figure 5, optical activatable probes are composed of an NIR fluorescent
molecule and a quenching molecule attached in close proximity by a linker that can be cleaved
only by the target enzyme. If exposed to excitation light in its native state, the probe is spectrally
silenced by the quencher. When the probe encounters its target, enzymemediated cleavage of the
linker releases the inhibiting quencher, effectively turning on the probe. Activatable NIR
probes have been used to visualize the activity of several enzymes, including cathepsin B and D,
HIV protease, matrix metalloproteinase (MMP)2 (Fig 6), protein kinase A, caspases, and
thrombin. An MR imaging activatable probe, named Egad Me, has also been designed and
consists of a caged Gd3_ ion that is undetectable in its native state because the cage prevents
water molecules from accessing the paramagnetic core, a requirement for T1 signal
enhancement. However, one wall of the cage is a galactopyranose ring that can be cleared by the
enzyme _-galactosidase. Probe activation is thereby mediated by galactosidase through opening
of the cage, allowing water molecules to enter and Gd3_ to exert its T1-lengthening effect on
water molecule protons.
Indirect Imaging
Reporter gene imaging methods have been developed for nuclear, optical, and MR imaging.
Classification of reporter systems can also be based on whether the reporter gene product is an
enzyme, receptor, or transporter (Table 3, Fig 3b). The most common enzyme based system uses
herpes simplex virus type 1 thymidine kinase (HSV1-tk) or its mutant derivative (HSV1-sr39tk)
as the reporter gene product, which enables imaging through phosphorylation and subsequent
intracellular entrapment and accumulation of radio labeled acycloguanosine (e.g. ganciclovir,
penciclovir) or pyrimidine nucleoside derivatives. The thymidine kinase of herpes simplex virus
type 1 is used because it can phosphorylate acycloguanosine derivatives, whereas the
mammalian version, which is more specific in its activity, cannot. In the exploitation of this
difference in substrate specificity, background noise is minimized. HSV1-tk has the added
advantage of also being a therapeutic gene in its ability to convert ganciclovir, a pro drug, into a
cytotoxic compound that inhibits DNA synthesis. This strategy, called suicide gene therapy, has
been actively pursued in oncology.
Reporter gene imaging has been used to study a variety of molecular and cellular processes such
as cell trafficking, gene delivery methods, endogenous gene expression, signal transduction
pathways, and gene expression regulation. For example, the p53 gene, the most commonly
mutated gene known in human cancer, encodes for a key transcription factor that halts cell cycle
progression in response to DNA damage. Dubrovnik et al studied p53 regulation of gene
expression by placing a dual reporter gene encoding for HSV1-tk and green fluorescent protein
under the control of a p53-specific enhancer, and the resulting construct was subsequently
transuded into tumor cells in rats. DNA damageinduced up regulation of p53 transcriptional
activity was then imaged by PET through expression of HSV1-tk and accumulation of its

reporter probe and confirmed by fluorescence microscopy of green fluorescent protein

expression. Although clinical application of this technology is limited by the need to introduce
reporter gene constructs into target tissue, reporter gene imaging promises to play a vital role in
the development of human gene therapy. Gene therapy has been significantly hampered by the
inability to effectively evaluate the delivery and expression of therapeutic genes. Various genetic
engineering strategies have been developed to link the expression of therapeutic genes with that
of reporter genes, allowing for in vivo reporter gene imaging to be used to monitor the location,
magnitude, and duration of therapeutic gene expression. Indeed, reporter gene imaging has been
used extensively in animal models of gene therapy, and initial studies in humans have also been
completed.