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Molecular Activity
Measurement Applications

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.

NIR imaging window graph

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.

Bone Imaging

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

IRDye® 680 BoneTag™ is an optical imaging probe that aids in bone remodeling and structural imaging studies. Together with another IRDye 800CW labeled targeting agent, two-color imaging on the Pearl® Trilogy Imaging System aids in localizing the area of disease.

BoneTag in mouse legs and spine

Figure 1. IRDye 800CW BoneTag was used to demonstrate resolution of structural imaging. Image captured with the Pearl Imaging System.

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.

visible blood vessels in mouse

Figure 2. Athymic male nu/nu mouse (~5-6 wks old), 0.5 hr after injection of IRDye 800CW PEG (1 nmole). Surface blood vessels are visible. Note: ability to visualize vasculature is dependent on mouse model used. Image captured with the Pearl Imaging System.

visible blood vessels and tumor in mouse

Figure 3. (A) Athymic male nu/nu mouse, ~4 hr after injection of IRDye 800CW PEG (1 nmole). Large blood vessels and tumor are visible. (B) High resolution (85 µm) image of the tumor region shows large blood vessels recruited to feed the tumor. Sequestration of contrast agent in tumor is likely due to enhanced permeability and retention. Image captured with the Pearl Imaging System.

800CW PEG detecting tumor

Figure 4. Athymic male nu/nu mouse, ~9 hr after injection of IRDye 800CW PEG (1 nmole). Tumor is clearly defined. Image captured with the Pearl Imaging System.

800CW PEG in mouse

Figure 5. Athymic male nu/nu mouse, minutes after receiving IRDye 800CW PEG (~0.1 nmole) intradermally on the tail (right side). Image highlights use of IRDye PEG as a lymph imaging agent. Image captured with the Pearl Imaging System.

Transfecting Cells for Molecular
Activity Measurement

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 Imaging

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).

tumors in mice

Figure 6. Mice were implanted with cells that constitutively express luciferase. After 2 weeks, mice received D-luciferin injections. Image captured with the Pearl Imaging System.

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.

fluorescent protein expression in mouse

Figure 7. Mouse xenografts from iRFP-22Rv1 or 22Rv1-luc/PN2 constitutively expressing cells were prepared (construct courtesy of Dr. Michael Henry, University of Iowa). IRDye 800CW EGF probe (green) was administered intravenously (1 nmole) for detection of EGF expression in vivo. Images for iRFP (red) and IRDye 800CW EGF expression were captured 48 hrs post injection while bioluminescence (blue) was captured 10 min post D-luciferin injection. Panels B, C, and D show individual images captured in the 700 nm, 800 nm, and bioluminescence channels respectively, while panel A presents a composite image for all three channels. Image adapted from Henry et. al. (2005) 5

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