Article Category: IRDye Reagents

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.

References:

  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.

Use Near-Infrared Fluorescent Probes for Pharmacokinetics and Biodistribution Studies

In Vivo Imaging with NIR Fluorescent ProbesNon-invasive preclinical imaging methods are critical for development of imaging agents and targeted therapeutics. Pharmacokinetics is the study of what the body does to a drug with respect to biodistribution and clearance. Traditionally-used radiolabeled probes have limitations such as cost, access, and safety. Near-infrared (NIR) fluorescence imaging offers a powerful alternative to radiolabeled probes for pharmacokinetics and biodistribution studies. NIR fluorescent optical imaging agents can be used to image the whole animal over time. And, more than one agent can be tracked in the same animal if each agent is labeled with a spectrally-distinct fluorophore.

In this webinar, Dr Amy Geschwender examines several case studies from the literature, and discusses:

  • Why NIR fluorescent probes are widely used for in vivo imaging
  • How fluorescence imaging of excised tissues and tissue sections is used to examine biodistribution in more detail
  • How to measure serum half-life and % injected dose per gram with NIR fluorescent probes


This webinar features data from the Pearl® Small Animal Imaging System, which was recently honored by Frost & Sullivan, in addition to advancements in NIR technology. Click here to learn more about this award.

Visit our website to learn more about BrightSite™ Optical Imaging Agents and IRDye® infrared dyes that can be used for your pharmacokinetic and biodistribution studies.

Journal Articles Citing Use of Odyssey® or Pearl® Imaging Systems and Near-Infrared Fluorescence

The following are 4 journal references citing the use of either Odyssey or Pearl Imaging Systems.

Affibody-DyLight Conjugates for in vivoAssessment of HER2 Expression by Near-Infrared Optical Imaging.

Zielinski R, M Hassan, I Lyakhov, D Needle, V Chernomordik, A Garcia-Glaessner, Y Ardeshirpour, J Capala and A Gandjbakhche
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
PLoS ONE 7(7): e41016 (2012). doi:10.1371/journal.pone.0041016

The HER2/neu gene is overexpressed in ~20% of invasive breast carcinomas. in vivo assessment of HER2 levels would aid development of HER2-targeted therapies and perhaps assist in selection of appropriate treatment strategies. This study describes HER2-specific probes for in vivo monitoring of receptor levels by near-infrared (NIR) optical imaging. Affibody molecules were labeled with DyLight750 dye, and affinity and specificity were confirmed in vitro. in vivo, Affibody-DyLight probes accumulated in HER2-positive breast cancer xenografts, but not in HER2-negative xenografts.

Fluorescent images were acquired at different time intervals after probe injection.
Fluorescent images were acquired at different time intervals after probe injection. Mouse bearing BT-474 xenograft tumor was injected with 10 µg HER2-Affibody-DyLight750 conjugate. Images were acquired every second for 1 minute with Pearl Impulse Imager (LI-COR Biosciences). doi:10.1371/journal.pone.0041016.s004

Animals were imaged with a custom NIR fluorescence-lifetime imaging system. The Pearl® Impulse Imager (LI-COR Biosciences) was used to monitor real-time accumulation of the Affibody probe in HER2-positive tumors during very early time points. Probe was injected during image acquisition, and images were captured every second for 1 minute. Probe accumulation in the kidney first, followed by tumor accumulation. Tumor fluorescence could still be detected 5 days after probe injection. This Affibody conjugate is useful for preclinical monitoring of HER2 status, and may have clinical utility.


Disruption of Kv1.3 Channel Forward Vesicular Trafficking by Hypoxia in Human T Lymphocytes

AA Chimote, Z Kuras, and L Conforti
Departments of Internal Medicine and Molecular & Cellular Physiology, University of Cincinnati, Cincinnati, Ohio
Journal of Biological Chemistry 287(3): 2055-67 (2012) DOI 10.1074/jbc.M111.274209

In solid tumors, hypoxia decreases immune surveillance. Kv1.3 channels on T lymphocytes are down-regulated by an unknown mechanism, inhibiting T cell function. The authors hypothesize that changes in membrane trafficking cause reduced expression of Kv1.3 at the cell surface. On-Cell Western cell based assays (Odyssey® Imager, LI-COR Biosciences) were extensively used to measure cell surface expression of Kv1.3.

Chronic hypoxia decreased cell surface expression of Kv1.3 in Jurkat cells. Inhibition of protein synthesis, degradation, or endocytosis did not block this effect. However, inhibition of forward trafficking in the trans-Golgi with brefeldin A (BFA) prevented hypoxia-induced reduction of Kv1.3 cell surface expression. Confocal microscopy confirmed retention of Kv1.3 in the trans-Golgi. Quantitative fluorescent Westerns (Odyssey Imager) demonstrated that expression of AP-1, which is required for clathrin-coated vesicle formation, is downregulated by hypoxia. These data indicate that chronic hypoxia disrupts clathrin-mediated forward trafficking of Kv1.3, thereby reducing immune surveillance by T cells.


Sequential Application of Anticancer Drugs Enhances Cell Death by Rewiring Apoptotic Signaling Networks

M Lee, A Ye, A Gardino, A Hheijink, P Sorger, G MacBeath, and M Yaffe
Dept of Biology, David H. Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA.
Cell 149:780-794 (2012). doi: 10.1016/j.cell.2012.03.031

Historically, standard treatments for human malignancies have been single drug therapies that cause DNA damage. Systems-based approaches and network analysis are now being used to examine how signaling can be re-wired by drug treatments that target dynamic network states. This study suggests that the timing and order of administration of certain drug combinations increases treatment effectiveness. Lee et al. pre-treated cells with epidermal growth factor receptor (EGFR) inhibitors, prior to DNA-damaging chemotherapy drugs.

Pre-treatment with erlotinib (an EGFR inhibitor) sensitized triple-negative breast cancers (TNBCs) to the DNA damage agent doxorubicin, and cell death increased by nearly 500%. Sensitization occurred only if the drugs were given sequentially. Transcriptional, proteomic, and computational analysis of signaling networks showed that dynamic network re-wiring was responsible for sensitization. Quantitative Westerns (Odyssey Imager; high-density, 48-sample blots) were used to monitor systems-level signaling dynamics. Erlotinib treatment made cells more susceptible to DNA damage by reactivating an apoptotic pathway that had been suppressed.


Investigation of Ovarian Cancer Associated Sialylation Changes in N-linked Glycopeptides by Quantitative Proteomics

V Shetty, J Hafner, P Shah, Z Nickens, and R Philip
Immunotope, Inc., Doylestown, Pennsylvania, USA
Clinical Proteomics 9:10 (2012) doi:10.1186/1559-0275-9-10.

CA125 is currently used as a biomarker for ovarian cancer, but is ineffective for detection of early stage disease. Previous research indicates that the level of sialic acid in total serum of ovarian cancer patients is elevated. Based on that idea, the authors suggest using N-linked sialyated glycopeptides as potential targets for early stage ovarian cancer biomarker discovery.

Shetty et al. used Lectin-directed Tandem Lableing (LTL) and iTRAQ quantitative proteomics to investigate N-linked sialyated glycopeptides, and identified 10 that were up-regulated in serum from ovarian cancer patients. Quantitative Western blot analysis of lectin-enriched glycoproteins (Odyssey Imager) was used to confirm the proteomic analysis. In ovarian cancer, increased sialylation of haptoglobin, PON1, and Zinc-alpha-2-glycoprotein was observed. Cancer-specific sialylation of glycopeptides may be a target for biomarker discovery.


Check out some of our Publications Lists for:

Click Chemistry Reagents from LI-COR® for Biomolecule Labeling

Biomolecule labeling continues to be a cornerstone feature of many in vitro and in vivo biological experiments. Click Chemistry has recently emerged as a convenient, versatile, and reliable method for labeling a wide variety of molecules for applications ranging from biomarker isolation to assay development.
Click Chemistry Workflow
LI-COR now offers a portfolio of Click Chemistry reagents for copper-catalyzed and copper-free methods. These products offer researchers flexibility to choose the correct reagent for a diverse array of applications. LI-COR Click Chemistry reagents include IRDye® 800CW, IRDye 680RD, and IRDye 650 infrared fluorescent dyes labeled with DBCO, azide, or alkyne groups.

Click Chemistry utilizes pairs of reagents that exclusively react with each other and are effectively inert to naturally-occurring functional groups such as amines. Unlike affinity interactions such as streptavidin-biotin, Click Chemistry forges covalent bonds between the reacting partners to deliver stable bioconjugates.

Click Chemistry reactions can be categorized into two separate groups, copper-catalyzed or copper-free. Copper-catalyzed Click Chemistry is used for initiating reactions between azides and alkynes. These reactions are also known as Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC). Although they initiate and accelerate Click Reactions, copper catalysts are cytotoxic and inappropriate for use in living systems.

Watch this informative webinar on IRDye Infrared Dye Reagents for Click Chemistry.

Click Chemistry Reagents Labeled with DBCO Groups Allow for Copper-Free Biomolecule Labeling Reactions

LI-COR now offers Click Chemistry reagents for copper-catalyzed and copper-free methods. One group of products within this portfolio includes IRDye® infrared dyes labeled with DBCO groups, which can be used for copper-free methods.

The dibenzocyclooctyne group (DBCO) allows copper-free Click Chemistry to be done with live cells, whole organisms, and non-living samples. DBCO groups will preferentially and spontaneously label molecules containing azide groups (—N3). Within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are naturally present in many biomolecules. Reaction of the DBCO group with the azide group is significantly faster than with the sulfhydryl group (—SH, thiol).
Click Chemistry Copper-Free Reaction

Click chemistry reagents with DBCO groups are available in 0.5 mg and 5 mg sizes for 3 dyes: IRDye 80CW, IRDye 680RD, and IRDye 650. IRDye 800CW DBCO also comes in a 50-mg pack size. For other sizes, contact LI-COR Custom Services.

Watch this 18-minute webinar to learn more about Click Chemistry applications and the new LI-COR® Click Chemistry reagents.

Create a Complete Molecular Imaging Workstation

pearltrilogybuildsystemCombining the Odyssey® CLx Infrared Imaging System with the Pearl® Small Animal Imaging System creates a versatile molecular imaging workstation for in vivo and in vitro imaging.

BrightSite™ Optical Imaging Agents or probes developed using IRDye® infrared dyes can be used for in vitro, in vivo, and tissue imaging. This technology offers researchers the ability to take research from the cell to the animal, all within one lab.

Odyssey CLx Infrared Imaging System Capabilities:

  • Cell-based assays (binding capacity, specificity, competition, etc.) for optical agent development
  • Histology and whole organ imaging for studying clearance and specificity
  • Simultaneous two-color detection for two targets or one target with sample normalization

Pearl Small Animal Imaging Capabilities:

Validation Workflow and Molecular Imaging WorkstationFigure 1. Validation and Use of an IRDye Fluorescent Probe. After probe labeling, in vitro cellular assays and microscopy are used to confirm specificity. The desired target is then imaged in animals. Excised organs and tissues> can be examined for more detailed localization of the probe. Animal image captured with Pearl Imaging System. A more comprehensive discussion of approaches for the development of fluorescent contrast agents has also been published. Reference: Kovar, et al. Anal Biochem 367(2007) 1-12.

Molecular imaging – achieved with near-infrared fluorescent technology from LI-COR!

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
Secondaries
IRDye 680RD
Secondaries
IRDye 680LT
Secondaries
Western Blot
In-Cell Western™ Assay Not Recommended
On-Cell Western Assay Not Recommended
Protein Array
Immunohistochemistry
Microscopy
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!

Use IRDye® Labeled Oligonucleotides for Safer, Faster Fluorescent Gel Shift Assays

The EMSA (electrophoretic mobility shift assay) is used to study protein:DNA complexes and interactions. Protein:DNA complexes migrate more slowly than unbound linear DNA on a non-denaturing gel, causing a “shift.”

Also called “gel shift” or “gel retardation” assays, EMSA can be used to analyze sequence-specific recognition of nucleic acids by proteins.

Traditional, radioactive EMSA protocols can be easily adapted to near-infrared fluorescence EMSA detection by using IRDye end-labeled oligonucleotides and imaging with the Odyssey® CLx or Odyssey Classic Infrared Imaging System, providing a safe and sensitive alternative.

Comparison of Detection Methods for Fluorescent Gel Shift Assay

For more information on the EMSA workflow and a sample protocol for infrared fluorescent mobility shift assays, visit our website.

In-Cell Western™ Assay Application: Response of COS-7 Cells to Hydroxyurea

Application: Detecting phospho-p53 in COS cells in response to Hydroxyurea


Example of In-Cell Western Assay: Effects of Hydroxyurea on phospho-p53 on COS-7 cells

In this In-Cell Western assay application, the response of COS-7 cells to increasing doses of hydroxyurea was measured by a specific antibody (Anti-phospho-p53 from Cell Signaling Technology, P/N 9286) that detects phosphorylated-p53 (Ser16). Total ERK1 was used for normalization. The image represents a 96-well two-color In-Cell Western with the 700 and 800 nm channels detecting phosphorylated-p53 (Ser16) and total ERK1, respectively. Background wells were incubated with secondary antibody but no primary antibody. IRDye® 680RD secondary antibodies were used for detection in the 700nm channel and IRDye 800CW secondary antibodies were usd for detection in the 800nm channel.

Dose response graph of % induction of p53 phosphorylation with hydroxyurea in COS-7 cells

The graph represents the average of four sets of quantitative data, demonstrating the percent induction of phosphorylated-p53 (Ser16). Plate-based assays such as this can be imaged on the Odyssey® CLx or Odyssey Sa Infrared Imaging System.

For more uses of In-Cell Westerns Assays, visit our website.

Now Use the Same Primary Antibody for Immunoprecipitation AND Western Blotting!

Quick Western Kit - IRDye 680RD, PN 926-68100

LI-COR® has just rolled out a new way that the recently-released Quick Western Kit – IRDye® 680RD (see Would you like to save at least 90 minutes the next time you do a Western blot?) can be used in your research.

Not only can the Quick Western Kit reduce Western blotting time by 90 min, the kit ALSO serves as a detection solution for post-immunoprecipitation samples by Western blot because it does not bind to denatured mouse monoclonal or rabbit monoclonal antibodies. The key benefit is the ability to use the same antibody for immunoprecipitation and post-immunoprecipitation detection by Western blot. Seriously, how cool is that!!??!!

Using the same primary antibody for IP and Western blotting with the Quick Western Kit

Figure 1. A431 cell lysates were immunoprecipitated overnight with a monoclonal antibody against p53. The resulting immunoprecipitates were separated by SDS-PAGE. Lane 1: Negative IP control; Lane 2: Test sample ; Lane 3: A431 cell lysate positive control. Western blotting was performed using the same p53 monoclonal antibody and incubated with IRDye 680RD Immunoprecipitation Detection Reagent.

Protocol: Detection of post-immunoprecipitation proteins by Western blot using the Quick Western Kit – IRDye 680RD

For more information, visit our website. Here’s the kit pack insert. To order this product (online ordering available in select countries), go to our ecommerce site.