Article Category: Pearl Small Animal Imagers

Visualizing Excised Tumor Samples with IRDye Fluorescent Dyes and the Pearl Trilogy

For cancer surgery to be considered fully successful, a tumor must be completely removed with no diseased tissue left behind. Tumor margin analysis in excised tissue samples is a widely-used assessment of whether a tumor was fully removed. Traditionally, margin analysis has been based on subjective evaluations of tissue differences in white light. In many cases, however, differences in cancerous and non-cancerous tissue are very difficult to discern in white light. For some cancers like head and neck squamous cell carcinoma, “positive margin” rates – rates of cancerous tissue (likely) being left behind – are as high as 40% [1].

Novel methods are needed to reduce positive margin rates and provide better clinical outcomes. Fluorescent dyes conjugated to tumor-specific monoclonal antibodies are emerging as a visual aid for tumor margin analysis and are proving to be particularly useful in cancers where positive margins still dominate clinical outcomes.

Evaluating Tumor Margins in Excised Tissue Samples With IRDye® 800CW-Cetuximab Fluorescent Images

Previous blog posts (Optical Probe Specificity and Dual-Modality Labeling with IRDye Near-Infrared Fluorescent Dyes and The Pivotal Role of Validation in Optical Probe Development) have highlighted in vivo and in situ clinical applications of IRDye 800CW-cetuximab. In an article published Rosenthal in Clinical Cancer Research, fluorescent contrast agents were shown to also improve visualization of cancer margins in excised tissue samples.

In this first-in-human study, 12 patients scheduled to have squamous-cell carcinoma tumors removed from the head or neck were given an infusion of IRDye 800-cetuximab prior to surgery. Fresh tumor tissue sections were imaged ex vivo with the LI-COR Pearl® Impulse imager to “determine the ability of tumor fluorescence to differentiate tumor from normal tissue and identification of positive margins” [1]. Like the intraoperative in situ results, the histopathological ex vivo results were also promising. The authors noted that “Fluorescence in histologically confirmed tumor tissue was significantly greater (P<0.001) than negative epithelial margins, muscle, and skin for each dose” [1].

Rosenthal and colleagues also utilized the Odyssey® imaging platform (LI-COR Biosciences) to quantify fluorescence in slide-mounted tissue sections after imaging with Pearl Impulse imager within the surgery suite. The fluorescent images taken in the Odyssey imager were correlated with routine H&E (hematoxylin and eosin) stains to compare IRDye 800CW-cetuximab against established pathological standards, corroborating the results of the Pearl Impulse scans.

The authors concluded “Here we demonstrate for the first time that … cetuximab-IRDye 800CW can be safely administered as a tumor-specific contrast agent,” and that “The use of real-time fluorescence imaging during ablative procedures to delineate tumor margins has the potential to reduce morbidity, improve locoregional control and reduce operative time “[1].

Cetuximab-IRDye 800CW in the Clinic Part 2: Enhanced Pathological Assessment with Fluorescent Probes

In a 2016 article published in The Journal of Pathology: Clinical Research, Warram utilized IRDye 800CW-cetuximab to address the lack of “tools to consistently discriminate tumor and normal tissue in real-time” for pathological assessments of tumor margins in head and neck squamous cell carcinoma (HNSSC) [2]. In this proof-of-principle study, the authors tested fluorescent assessment of diseased tissue margins against standard histological methods. 80 tumor margin assessments were collected from post-resection wound beds of 20 mice with SCC1-luc tumors after administration of IRDye 800CW-cetuximab.

The results were significant: fluorescent images improved pathologist prediction of positive tumor margins from 21/39 (49%) to 33/39 (85%), or a 36% increase in sensitivity in positive tumor margin predictions. The authors noted false negative margin predictions lead to a 90% 5-year post resection mortality rate, demonstrating the magnitude of impact that fluorescent-guided tumor margin analysis may have on patient outcomes.

Figure 1: Fluorescent analysis of primary tumor specimen [2]. Circles represent positive or negative biopsy-confirmed cancer cells, showing the distribution and specificity of IRDye 800CW to diseased cells.
Figure 2: Demonstration of how specificity translates into margin classification in excised tissue samples [2].

The authors ultimately concluded that “The ability of fluorescence assessment to localize diseases in these margins was sensitive and specific with a NPV of 87%, which was superior to both surgical assessment (58%) and pathological assessment (66%)” [2]. The authors also noted that “This report provides evidence that tumor-specific fluorescence can be used by the surgeon or pathologist to guide sampling for frozen sections” [2]. Although the current research does not suggest that fluorescence is a bona fide replacement for current methods, “Fluorescence-guided pathology can … be easily implemented into the clinical care workflow and used in adjunct to fluorescence-guided surgery to help guide the pathologist when assessing margins for both intraoperative assessment and staging” [2].


In certain cancers like head and neck squamous cell carcinoma, even the most effective treatment still has relatively high rates of failure. Novel methods are needed to reduce the failure rate and provide better clinical outcomes.

Fluorescent dyes conjugated to tumor-specific monoclonal antibodies are emerging as a promising visual aid for tumor analysis. Rosenthal and Warram showed how fluorescent dye-antibody conjugates can enhance tissue assessments, also demonstrating the versatility of fluorescent probes for both in situ and in vitro assessments.

For more exciting clinical applications of IRDye probes and conjugates, visit the Optical Probe Development and Molecular Activity Measurement web pages.

Do you think IRDye infrared dye-labeled probes could be used in your research? Let us help! Contact LI-COR Custom Services.


  1. Rosenthal, E.L., et al. Safety and Tumor-specifity of Cetuximab-IRDye800 for Surgical Navigation in Head and Neck Cancer. Clin Cancer Res 2015, Aug; 21(16):3658-3666. doi: 10.1158/1078-0432.CCR-14-3284.
  2. Warram, J. M., de Boer, E., van Dam, G. M., Moore, L. S., Bevans, S. L., Walsh, E. M., & Young, E. S., (2016, March 2). Fluorescence Imaging to Localize Head and Neck Squamous Cell Carcinoma for Enhanced Pathological Assessment. Journal of Pathological Cancer Research, 2(2), 104-112. doi:10.1002/cjp2.40

Use of IRDye Infrared Dye-Labeled Optical Probes for Intraoperative Tumor Visualization

A major challenge in cancer surgery is being certain that all the tumor has been removed, including the residual cancer cells not immediately identified with the naked eye after resection. Surgeons need intraoperative methods of imaging tumors to assist them in identifying healthy and diseased tissue. These methods need to be safe and effective. Near-infrared (NIR) fluorescent optical probes may provide a viable solution.

Near-infrared fluorescent optical probes have been used intraoperatively in clinical trials. These NIR dye-conjugated compounds offer several advantages for use in the operating room. NIR probes can be used safely, unlike other imaging modalities that require radiation (such as CT, PET, and SPECT).

IRDye® dye-conjugated optical probes have been shown to be sensitive and biomarker-specific and their fluorescent signal correlates with tumor location observed by other imaging methods and traditional pathology. Because fluorescence from NIR optical probes is invisible to the human eye, visualization of the surgical field of view with white light is unimpeded.

Fluorescence from tissue excised during surgery can be visualized while in the operating room and used to assess whether resection of the tumor is complete. Traditional pathologic examination can then be done for confirmation. Specialized NIR imaging equipment, such as the Pearl® Imaging System, has been used successfully to image tumor sections during an operation.
The following two studies involved the intraoperative use of near-infrared fluorescence optical probes.

IRDye 800CW Dye-Conjugated Probes Provide Verification of Tumor

In this study by van Driel, et al., investigators evaluated the Artemis imaging system, developed in collaboration with the Center for Translational Molecular Medicine. The goal of the study “was to evaluate the Artemis camera in two oncological procedures in which real-time NIR fluorescence could be of added value: (a) radical tumor resection; and (b) detection of sentinel lymph nodes. . .” [1]
For the evaluation of the Artemis imaging system, the investigators used ICG and two IRDye® 800CW infrared dye-conjugated nanobodies. “IRDye 800CW (LI-COR, Lincoln, NE, USA, λex=774 nm, λem=789 nm) was chosen because it is one of two novel fluorophores in the process of clinical translation.” [1] The study assessed the sensitivity and utility of the Artemis system for intraoperative detection of head-and-neck tumors and sentinel lymph nodes in xenograft mouse models. [1]

Fluorescent images were concurrently acquired with the Pearl® Impulse Small Animal Imager (LI-COR). [1] “The Pearl system is expected to be an order of magnitude more sensitive than the Artemis, and therefore, these images serve as a ground truth comparison.” [1]

IRDye 800CW Dye-Labeled Probes Target VEGF and HER2

Research performed by Terwisscha van Scheltinga, et al. used IRDye 800CW dye-labeled antibodies to investigate their use in targeting certain tumors for optical surgical navigation [2]. The group concluded that “NIR fluorescence-labeled antibodies targeting VEGF or HER2 can be used for highly specific and sensitive detection of tumor lesions in vivo. These preclinical findings encourage future clinical studies with NIR fluorescence–labeled tumor-specific antibodies for intraoperative-guided surgery in cancer patients.” [2]

In this preclinical mouse study, fluorescent optical imaging with IRDye 800CW NHS ester coupled to bevacizumab was compared to PET imaging with 89Zr (5 MBq)-labeled bevacizumab or trastuzimab along with a non-specific antibody control, 111In-IgG (1 MBq). [2]

The researchers of this study stated, “IRDye 800CW is a NIR fluorophore with optimal characteristics for clinical use, allowing binding to antibodies when used in its N-hydroxy-succinimide (NHS) ester form. A preclinical toxicity study with IRDye 800CW carboxylate showed no toxicity in doses of up to 20 mg/kg intravenously or intradermally.” [2] They concluded that “In a preclinical setting, NIR fluorescence–labeled antibodies targeting VEGF or HER2 allowed highly specific and sensitive detection of tumor lesions in vivo.” [2]

IRDye 800CW dye-conjugated optical probes are currently involved in over a dozen clinical trials for a wide range of different cancers. These studies demonstrate the use of IRDye probes for optical surgical navigation. Several studies have employed the use of dual-labeled probes showing the strength of combining near-infrared fluorescence with other imaging modalities.

Examples of optical probe applications are detailed on Optical Probe Development and Molecular Activity Measurement web pages.

Do you have questions about how IRDye infrared dye-labeled probes could be used in your research or need help conjugating your optical probe? If so, please contact LI-COR Custom Services.


  1. van Driel, P.B.A.A., et al. Characterization and Evaluation of the Artemis Camera for Fluorescence-Guided Cancer Surgery Mol Imaging Biol (2015) 17:413Y423. doi: 10.1007/s11307-014-0799-z
  2. Terwisscha van Scheltinga, A.G.T., et al. Intraoperative Near-Infrared Fluorescence Tumor Imaging with Vascular Endothelial Growth Factor and Human Epidermal Growth Factor Receptor 2 Targeting Antibodies J Nucl Med 2011; 52:1778–1785. doi: 10.2967/jnumed.111.092833.

Create a Customized List of Journal Articles that Reference LI-COR Imaging Systems

Finding out how other researchers have used LI-COR® imaging systems and reagents can really help when you are trying to decide on which system is best for your lab. With over 10,000 journal citations, the Odyssey® Imaging Systems have a long, proven track record in life science research.

There is now a tool that you can use to customize a list of peer-reviewed references specific to your research and application interests. You can access this new Publications Database through pages on our website, such as Products > Imaging System pages (for example, Odyssey CLx > Who’s Using it) and Application pages (for example, Quantitative Western Blots > Publications) or through the link in the footer, which is on the bottom of all web pages.

You can filter results by four categories. Select at least one filter. Each filter you select narrows the search by making the resulting set a combination of all filter parameters.

Let’s go over the various options you can use to create your customized publications list.

You can filter by Research Area. The list of research areas includes 72 different categories. Choose a single, or multiple, area(s). Remember, the more you choose, the narrower your search will be.

You can filter by Instrument.

You can filter by Application.

You can filter by Country. This is the country of the corresponding author’s email address.

For example, if you select “Apoptosis” in Research Area, “Odyssey CLx” in Instrument, and “Germany” in Country, you will receive a list of three publications that specifically reference apoptosis, the Odyssey CLx, and publications where the corresponding author is from Germany.

You can sort the columns in the results by clicking on the corresponding header. You can also show 10, 25, 50, or all entries. If the list does not suit your needs, you can Clear All Filters and start over.

If the number of publications returned is large, you can refine the set using keywords, such as your protein or disease of interest, separated by a space. Results displayed will contain all the terms in the search field. You can also use the digital object identifier (DOI) prefix unique to each publishing group to search for publications in specific journals. For example, use 10.1074 for the Journal of Biological Chemistry and other ASBMB journals.

We regularly add publications to the database, so check back frequently to see who is using LI-COR imaging systems to get published, and how they are using the instrument in their research.

Create your own customized publication list and, if you have feedback on how to improve this tool, please click on the Feedback button and let us know!

No-Hassle Near-Infrared Fluorescent and Bioluminescent Optical Imaging

Pearl Trilogy Workstation
The Pearl® Trilogy Small Animal Imaging System is a simple, economical way for your lab to begin in vivo optical imaging. Starting at $65,000 (US List), the Pearl Trilogy offers affordable small animal near-infrared fluorescent and bioluminescent optical imaging.

Combine the Pearl Trilogy with the Odyssey® CLx Imager and create a complete workstation that allows you to go from in vitro to in vivo to ex vivo using the same trusted technology and reagents.

The revolutionary FieldBrite™ Xi2 technology approach to imaging in the Pearl Trilogy Imager allows you to detect smaller and deeper targets accurately in a single acquisition. You don’t need to worry about saturation or having to adjust images.

FieldBrite Xi2 is specifically optimized for small animal imaging, ensuring that you get the highest quality data possible. FieldBrite Xi2 technology offers:

  • Uniform Illumination
  • Excellent Sensitivity
  • Wide Dynamic Range

Bioluminescent Optical Imaging

Figure 1. Bioluminescent detection of subcutaneous 4175 (LM2) luc + human triple negative breast cancer cell line in athymic NCR nu/nu mouse.

Image courtesy of Michael Chiorazzo, Elizabeth Browning and Jim Delikatny, Small Animal Imaging Facility, University of Pennsylvania.

Ask for a quote today. Get your lab up and running quickly – it’s easy to use so little training will be needed for even novices. And, you can count on the reliable data for your in vivo imaging research from the Pearl Trilogy Small Animal Imaging System.