<b>Small Animal Optical Imaging: Seeing Beyond The Visible With Infrared Dyes</b><br/><br/>

Small Animal Optical Imaging: Seeing Beyond The Visible With Infrared Dyes

by Harry Osterman and Amy Schutz-Geschwender

Optical imaging offers the potential for non-invasive study of molecular targets inside the body of the living animal. This technology has been used to follow the progression of disease, the effects of drug candidates on the target pathology, the pharmacokinetic behavior of drug candidates, and the development of biomarkers indicative of disease and treatment outcomes. Currently, the three major types of labels used in optical imaging are bioluminescence, fluorescent proteins, and fluorescent dyes or nanoparticles. Bioluminescence and fluorescent proteins require engineering of cell lines or transgenic animals that carry the appropriate gene. Because fluorescent dyes do not have this requirement, they have the potential to translate to clinical applications. For example, the carbocyanine dye indocyanine green (ICG; also known as Cardiogreen R), has been used in the clinic for over 25 years as a dilution indicator for studies involving the heart, liver, lungs, and circulation.⁽¹⁾

Figure 1. Molar extinction coefficient characterization of water, hemoglobin and oxyhemoglobin (400 - 1000 nm). Click to enlarge.

Near-infrared (NIR) fluorophores minimize the optical challenges of detecting photons in tissues. A fundamental consideration in optical imaging is maximizing the depth of tissue penetration, which is limited by absorption and scattering of light. Light is absorbed by hemoglobin, melanin, lipids, and other compounds present in living tissue.⁽²⁾ Because absorption and scattering decrease as wavelength increases, fluorescent dyes and proteins absorbing below 700 nm are difficult to detect in small amounts at depths below a few millimeters.⁽³⁾In the NIR region (700-900 nm), the absorption coefficient of tissue is at its lowest (Figure 1) and light can penetrate to depths of several centimeters.⁽⁴⁾ Above 900 nm, light absorption by water begins to cause interference. Autofluorescence is also an important consideration. Naturally-occurring compounds in animal tissue can cause considerable autofluorescence throughout the visible spectral range up to ~700 nm, which can mask the desired signal.

A number of NIR dyes have been employed for in vivo imaging, including Cy5.5 and Cy7 (GE Healthcare, Piscataway, NJ), Alexa Fluor 680 and Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA) and IRDye 680 and IRDye 800CW (LI-COR Biosciences, Lincoln, NE). Cy5.5 has been used in the past primarily due to the lack of other dyes more suitable for imaging. The excitation/emission maxima for this dye (675 nm/694 nm) fall in the range affected by tissue autofluorescence, impacting its overall performance, and Cy5.5 has also been shown to cause higher background in cellular assays due to non-specific binding. In contrast, IRDye 800CW has excitation/emission maxima at 785 nm/810 nm, precisely centered in the region known to give optimal signal-to-background ratio for optical imaging.⁽⁴⁾Quantum dots, with their photostability and bright emissions, have generated a great deal of interest; however, their size precludes efficient clearance from the circulatory and renal systems and there are questions about their long-term toxicity.⁽⁵⁾ An alternative, the pthalocyanine dye IRDye 700DX, is substantially more photostable than Alexa Fluor 680 and Cy5.5 with the added potential to be used for photodynamic therapy in treatment of cancer.⁽⁶⁾

A number of studies have demonstrated the use of IRDye infrared dyes for optical imaging.

• In a comparison of gamma scintigraphy and NIR imaging, a cyclopentapeptide dual-labeled with ¹¹¹indium and IRDye 800CW was used to image α_νβ₃-integrin-positive melanoma xenografts.⁽⁷⁾ The tumor regions were clearly delineated by optical imaging of the IRDye 800CW signal. In contrast, tumor boundaries could not be identified by scintigraphy due to high noise levels.

• IRDye 800CW has been conjugated to epidermal growth factor (EGF) for imaging of tumor progression.⁽⁸⁾ In this longitudinal study, probe accumulation was monitored in orthotopically-implanted prostate tumors that over-express the EGF receptor. Fluorescence intensity correlated well with tumor size, and lymph node metastasis could be imaged upon endpoint dissection. Figure 2 shows an example of a SCID mouse bearing an orthotopic prostate tumor visualized with IRDye 800CW EGF.

• Cy5.5 and IRDye 800CW were used to label EGF and the effectiveness of these probes for in vivo imaging of breast cancer cell lines in subcutaneous tumors in mice was evaluated.⁽⁹⁾The study showed a significant reduction in background and an enhanced tumor-to-background ratio when IRDye 800CW was compared to Cy5.5, suggesting that longer-wavelength dyes may produce more effective targeting agents for optical imaging.

• Pamidronate has been labeled with IRDye 78 (an NIR fluorophore closely related to IRDye 800CW) and used as a bone imaging agent to detect osteoblastic activity in a living animal.⁽¹⁰⁾ IRDye 78-pamidronate had rapid clearance with an early half life of 5 minutes. Use of such optical agents to study skeletal development, osteoblastic metastasis and coronary atherosclerosis was proposed.

• In vivo imaging of prostate tumor xenografts has been performed using IRDye 78-labeled GPI, a potent inhibitor of PSMA (prostate specific membrane antigen⁽¹¹⁾). Both IRDye 78 and IRDye 78-GPI showed rapid biodistribution and clearance. The authors demonstrated sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells, as well as in vivo imaging of xenograft tumors.

• Use of human serum albumin labeled with IRDye 800CW (HSA800) as a tracking agent for mapping of sentinel lymph nodes was demonstrated using an intra-operative NIR fluorescence imaging system.⁽¹²⁾ HSA800 demonstrated good entry to lymphatics, flow to the sentinel lymph nodes, retention in the sentinel lymph nodes, fluorescence yield, and reproducibility.

The molecular imaging workflow

Figure 2. Tumor imaging with IRDye 800CW EGF. A SCID mouse bearing an orthotopic prostate tumor was injected with 1 nmole IRDye 800CW-EGF and imaged 72 hours post-injection.

The basic steps for making, validating and using an NIR fluorescent probe are summarized below. A more comprehensive discussion of approaches for the development of fluorescent contrast agents was published recently.⁽¹³⁾

Probe preparation

In vivo imaging projects typically begin with identification of a possible tracking agent or probe, such as a receptor ligand, peptide, small molecule, antibody or virus. Dyes with NHS ester reactive groups, such as IRDye 800CW NHS ester, can be used to label primary amines such as lysine residues to prepare the labeled probe. Kits are commercially available for labeling and purification of some of these targeting agents.

In vitro validation

Cell-based assays can often be used to evaluate binding and specificity in vitro before animal studies begin. A variety of approaches have been used for in vitro testing, including the "In-Cell Western" format.^{(8, 13)} In this assay, cultured tumor cells in microplates are incubated with the labeled targeting agent to assess binding. Specificity is evaluated by methods such as blocking access to the target with an antibody or competition with an excess of unlabeled agent. Fluorescence emission from each microplate well is then quantified.

In vivo clearance

Clearance studies with both the unconjugated dye and the labeled probe are important for accurate interpretation of imaging data. Signal may be nonspecifically retained in regions of the body that block or mimic the intended target (such as the liver, kidneys, or bladder), and could result in misinterpretation of data if these controls are not performed. Time courses of probe clearance also help to establish the optimal time for imaging in subsequent experiments.

Imaging

The probe can then be used to image the desired target in animal studies. If possible, specificity should also be confirmed in vivo. One approach is to pre-inject the animal with an excess of unlabeled agent or other compound that blocks or competes with binding of the targeting agent.⁽⁸⁾

Tissues and organs

At the end of the imaging study, animals can be sacrificed and organs or tissues can be excised and imaged to confirm the presence of the probe in the desired location. Imaging of whole organs provides a quick and semi-quantitative estimate of signal intensity. For more detailed study, sections can be prepared from frozen or paraffin-embedded tissue and imaged at higher resolution.

About the authors

Harry Osterman is Director of Market Research at LI-COR Biosciences. He received a Ph.D. in biochemistry from Kent State University and was a postdoctoral fellow in molecular biophysics and biochemistry at Yale University. Amy Schutz-Geschwender is Director of Molecular Biology Research at LI-COR Biosciences. She received her Ph.D. in molecular and cellular biology from the University of Colorado at Boulder.

More information about infrared dyes and their benefits in various research applications is available from:

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