Measuring Molecular Activity
with Optical Imaging
Optical imaging modalities are ideal for observing and characterizing biological and cellular processes in vivo with the right probe. These imaging modalities include detection using bioluminescence as well as fluorescence. Molecular activity can be measured by optical imaging either directly, using fluorescence optical probes or indirectly, using cells transfected with luciferase gene or iRFP.
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NIR Optical Imaging Probes
Near-infrared (NIR) optical probes can be used to illuminate features within the animal (bone, vasculature, etc.). These probes may include specific targeting agents or non-specific contrast agents conjugated to near-infrared dyes.
Detection in the NIR spectrum (680 nm to 820 nm) is ideal for measurement of in vivo molecular activity as it provides several advantages:
Light absorbance by, and background autofluorescence from animal tissue is significantly lower in the NIR region. These properties allow better light penetration and higher signal-to-noise ratios for detecting deeper targets in vivo.
Compared to other imaging modalities, images can be captured within seconds, allowing rapid screening of mice.
Compared to other modalities, optical imaging is more economical.
What is Bone Imaging?
Bone imaging is commonly used to provide anatomical landmarks to localize an area of tumor or disease. For instance, skeletal structures can be visualized using a dye-labeled bone targeting compound, while a second tumor-targeting agent labeled with a spectrally distinct dye, can help localize the tumor to bone anatomy. The two images can be overlaid to better define the location of the tumor or disease. Additionally, bone imaging in bone remodeling studies can detect bone growth or bone loss due to disease.
"…Investigators in the Vanderbilt Center for Bone Biology are using that probe [developed in Manning lab] to look at TGF‑beta in bone, and in and around tumors that may be in the bone, and that's been very fruitful with the Pearl."
Dr. H. Charles Manning, Vanderbilt University
Bone Imaging using NIR Fluorescence
Vascular and Lymphatic Imaging
What is Vascular and Lymphatic Imaging?
The vascular endothelium in the tumor microenvironment is often discontinuous, which allows molecules to diffuse into the surrounding tissue.1,2 Lymphatic drainage in these regions is also poor.3 As a result, the tumor tissue is permeable to large molecules, and retains these molecules. Because of these properties, tumor vasculature and lymphatics can be studied using in vivo optical imaging probes.
Vascular and Lymphatic Imaging
using NIR Fluorescence
IRDye 800CW dye is detected in the NIR spectrum where background autofluorescence from tissues is low. As a result, high signal-to-noise ratios are generated and provide images with excellent quality and resolution.
A near-infrared dye-conjugated contrast agent, such as IRDye 800CW PEG (polyethylene glycol), serves as a non-specific contrast agent for vascular imaging. The labeled agent is administered to mice intravenously (IV injection) and highlights surface vasculature for 30 minutes post-injection (Figure 2). The retention of the agent is visible in the tumor 4 hours post-injection (Figures 3A, B; requires appropriate mouse model*) and the tumor region is defined by 9 hours post-injection (Figure 4).
*Success of vascular imaging depends on the mouse model used. Vessels may be less visible in mice that are obese or have hair.
IRDye 800CW PEG also serves as an effective lymph tracking agent and can be administered by intradermal injection (Figure 5). Other contrast agents labeled with IRDye 800CW have been used in intraoperative identification of lymphatic branches and small sentinel lymph nodes, and have outperformed NIR quantum dots.4
The Pearl Trilogy Imaging System allows visualization of vasculature and lymphatics, without the need to change exposure settings between scans. Watch this two-minute video on rapid time-lapse lymphatic and lymph node imaging.
Transfecting Cells for Molecular
Measuring Molecular Activity with Transfected Cells
Molecular activity can be measured using transfected cells or transgenic animals expressing either the luciferase gene (bioluminescence detection) or fluorescent proteins like iRFP (fluorescence detection). With specific promotors, these genes can be used to monitor levels of expression (either constitutive or inducible) within an animal in context of the disease being studied.
Bioluminescence detection is generally used for cell tracking and cellular expression studies. The substrate, luciferin, is required along with the luciferase-construct to complete the light emitting pathway. For example, cells that constitutively express the luciferase gene may be injected into a mouse and followed over time for cellular distribution. Likewise, cells that express the luciferase gene under an inducible promotor may be monitored through various chemical treatments to assess how the levels of expression change.
Bioluminescence Imaging on Pearl Trilogy
The Pearl Trilogy Imaging System can detect bioluminescence for in vivo molecular activity measurements (Figure 6).
Fluorescence Protein Expression
Infrared fluorescence protein expression can be used to measure cell tracking and cellular expression. Unlike bioluminescence, iRFP constructs do not require a substrate, but simply need to be excited using a light source. The emitted light is then detected by the imaging system.
Fluorescent Protein Detection on Pearl Trilogy
The Pearl Trilogy Imaging System can detect fluorescent protein expression in the 700 nm channel for in vivo molecular activity measurements.
- Vasey, PA, et al; Phase I Clinical and Pharmacokinetic Study of PK1
[N-(2-Hydroxypropyl)methacrylamide Copolymer Doxorubicin]: First Member
of a New Class of Chemotherapeutic Agents- Drug-Polymer Conjugates;
January 1999; Clin. Cancer Res. 5: 83-94.
- Matsumura, Y and H Maeda. A New Concept for Macromolecular Therapeutics in Cancer Chemotherapy: Mechanism of Tumoritropic Accumulation of Proteins and the Antitumor Agent Smancs; December 1986; Cancer Res 46: 6387-6392.
- Seymour, LW; Passive Tumor Targeting of Soluble Macromolecules and Drug Conjugates; January 1992; Critical Reviews in Therapeutic Drug Carrier Systems; 9(2): 135-187.
- Tanaka, E., Choi, H.S., Fujii, H. et al. Image-Guided Oncologic Surgery Using Invisible Light: Completed Pre-Clinical Development for Sentinel Lymph Node Mapping; Ann Surg Oncol (2006); 13:1671.
- Drake, J.M., Gabriel, C.L. & Henry, M.D. Assessing tumor growth and distribution in a model of prostate cancer metastasis using bioluminescence imaging. Clin Exp Metastasis (2005) 22: 674.