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.

NEW! IRDye® Goat Anti-Mouse IgM Secondary Antibodies from LI-COR®!

IRDye Dye-labeled Goat anti-Mouse AntibodiesOur IRDye secondary antibody line is growing! We have recently added IRDye Goat anti-Mouse IgM (μ chain specific) secondaries labeled with:

  • IRDye 800CW (PN 926-32280)
  • IRDye 680RD (PN 926-68180) or
  • IRDye 680LT (PN 926-68080).

Just like all of the LI-COR IRDye secondary antibodies, these are highly cross-adsorbed secondary antibody conjugates suitable for a variety of applications (see the table below).

IRDye 800CW secondary antibodies are the antibodies of choice for a wide variety of applications in the 800 nm channel (see the list below). IRDye 800CW secondary antibodies can be used for 2-color detection when multiplexed with IRDye 680RD or IRDye 680LT secondary antibodies.

IRDye 680RD secondary antibodies are the antibodies of choice for In-Cell Western Assay and Western blot applications in the 700 nm channel. These antibodies can be used for 2-color detection when multiplexed with IRDye 800CW secondary antibodies. These antibodies are our most universal use 700 nm channel antibodies. Start using IRDye 680RD first over other 700 nm dyes. Dilution working range 1:10,000 – 1:40,000.

IRDye 680LT secondary antibodies have been proven the brightest signal for Western blot detection in the 700 nm channel and are comparable to Alexa Fluor 680 secondary antibodies. Choose IRDye 680LT secondary antibodies to get high signal and for specific uses of detection in the 700nm channel. These antibodies are not recommended when getting up and running on system. Once established near-infrared protocols are optimized with IRDye 680RD, IRDye 680LT can be used to optimize signals in the 700 channel. Dilution range 1:20,000 – 1:40,000. Note: optimization may be required with IRDye 680LT.

Application IRDye 800CW
IRDye 680RD
IRDye 680LT
Western Blot
In-Cell Western™ Assay Not Recommended
On-Cell Western Assay Not Recommended
Protein Array
2D Gel Detection
Tissue Section Imaging
Small Animal Imaging Not Recommended
Virus Titration Assay Not Known Not Known
FRET-based Assay Not Known Not Known

Note: Now, as of December 15, 2014, you can also get 0.1 mg sizes of all of our IRDye dye-labeled secondary antibodies. Check out our complete listing here and our new filtering tool!