Article Category: In Vivo Imaging System and Applications

The Pivotal Role of Validation in Optical Probe Development

Underlying every successful clinical application of fluorescent probes is a rigorous, strategic probe validation process. Previous blog posts have discussed the importance of probe specificity, binding affinity, and distribution, and the validation process is where these and other parameters are determined. Given the time and expenses involved in clinical translation, efficient and accurate probe validation is essential.

A Systematic Approach to Developing and Validating Optical Imaging Contrast Agents

In a foundational 2007 article published in Analytical Biochemistry, Kovar demonstrated the principal steps involved in developing fluorescent optical probes suitable for human clinical use. The authors began with a comprehensive review of NIR fluorochromes, such as IRDye® 800CW, and targets and ligands for fluorescent optical probes, including monoclonal antibodies, tumor surface proteins, peptides, and small molecules. Steps for development and validation described in the remainder of the article are “applicable to any dye-conjugated optical agent,” demonstrating the versatility of this systematic approach [1].


The first step in probe development is the conjugation of target and NIR fluorochrome. In this study, the authors conjugated IRDye 800CW to five commercial epidermal growth factor (EGF) sources in equivalent ratios and evaluated signal intensity via the In-Cell Western™ (ICW) assay method. ICW imaging demonstrated variation between the signal strength of each EGF source. Because “Variations in signal strength measured in this fashion have the potential to predict probe performance in vivo,” choosing the correct target for conjugation is a critical first step [1].


Prior to animal imaging, probe specificity and binding affinity are validated in vitro. Here, the authors again chose the In-Cell Western (cytoblot) method to evaluate IRDye 800CW EGF for binding specificity. Specifically, PC3M-LN4 and 22Rv1 human prostate adenocarcinoma cells were cultured in microtiter plates and were “treated with serial dilutions of labeled EGF to verify a high affinity binding of EGFR-targeted dye” [1]. Specificity was then determined by blocking access of EGF to the EGF receptor with an anti-EGFR monoclonal antibody, and by competition with unlabeled EGF [1]. The authors concluded that “Characterization of the targeting agent in a cell-based assay can simplify probe development,” and that although “success in a cell-based assay format does not guarantee performance in vivo, failure at this step is generally predictive of failure in the animal” [1].

Learn more about the In-Cell Western Method.


Next, the authors validated probe specificity and clearance in vivo in living mice. This is the process of determining probe uptake by the target vs surrounding tissues, the rate at which the probe is expelled from the organism, and the probe to background ratio in the body of mice. First, clearance kinetics of unconjugated IRDye 800CW were established. Then, clearance measurements for IRDye 800CW-anti-EGFR antibody conjugates were established in mice bearing PC3M-LN4 subcutaneous or orthotopic tumors to ensure the conjugate did not accumulate non-specifically in the mouse. This interaction between clearance kinetics and specificity can impact in vivo analysis by falsely indicating tumor tissue in pooled optical probe in the liver or kidneys.


Lastly, the PC3M-LN4 tumors were excised and injected intravenously with IRDye 800CW EGF or pre-injected with C225 anti-EGFR monoclonal antibody prior to dosing with IRDye 800CW EGF for ex vivo analysis. After imaging, the distribution of the IRDye probe was assessed and fluorescence signal area was determined against a control, optical agent only, and C225 competition.

The authors ultimately concluded that “Fluorochrome-labeled molecular probes are valuable tools for non-invasive longitudinal study of tumorigenesis and metastasis, preclinical studies of the effects of therapeutic agents, and pharmacokinetic and pharmacodynamic studies of drug-target interactions” [1]. Since this paper was published over a decade ago, IRDye labeled molecular probes have been featured in more than 20 clinical trials around the world.

EGFR-Specific Optical Probes Improve EGFRvIII-Targeted Molecular Imaging

In a 2014 study published in Cancer Biology & Therapy, Gong demonstrated an application of structured probe validation. In this investigative study, the authors created and validated the specificity, binding affinity, distribution, and clearance of three EFGRvIII-targeted fluorescent optical probes. An EFGR-specific affibody, the therapeutic antibody panitumumab, and an EGF ligand were conjugated with IRDye 800CW to create three probes: Aff800, Pan800, and EGF800. The experimental target was rat glioma cell line F98, a known over-expresser of EGFR. A control assay contained EGFR expression-devoid F98 parent (F98-p) cells, and two experimental assays contained F98-derived transgenic cells expressing EGFR or EGFR-vIII. Each probe was compared with each cell-based assay and imaged for comparison, creating a total of nine experimental conditions.

Comparison of specificity and binding affinity between the experimental conditions was performed in cell-based assays using the In-Cell Western method. All three probes successfully bound to F98-EGFR, and Pan800 and Aff800 bound to F-98vIII. Signal intensity was also compared in the nine conditions to assess if binding was dose-dependent. The authors concluded “Little signal was detected when Aff800 and Pan800 were incubated with F98-p [the expression-devoid parent cells], indicating that their interactions with F98-EGFR and F98-vIII is highly specific” [2].

Next, probe target specificity to EGFR- and EGFRvIII-expressing tumors and clearance profiles were assessed in vivo. Mice with F98-p, F98-EGFR, and F98-vIII xenograft tumors were injected with the three probes and imaged with the Pearl® Impulse Small Animal Imaging System (LI-COR Biosciences). Fluorescent signal to background ratio for each of the nine probe-tumor conditions were assessed, again revealing highly specific interactions between Aff800 and Pan800 with F98-EGFR and F98-vIII expressing tumors. EGF-800 signal was high in F98-EGFR tumors, corroborating cell based assay results.

Lastly, tumor-containing organs were dissected and imaged ex vivo, validating the previously-measured fluorescence signals and assessing probe distribution in targets. This last step in validation was consistent with in vitro scans, again demonstrating Aff800 and Pan800 affinity to F98-EGFR and F98-vIII tumors. Based on these results, Aff800 and Pan800 may be valuable in “imaging of heterogenous tumors containing both versions of receptors” (EGFR, EGFRvIII) [2]. Alternatively, due to optimal clearance kinetics, Aff800 EGF800 is preferable in scenarios where imaging must be performed within a short time after probe administration” [2].


This example from Gong demonstrates how different optical probes may be used to assess different tumor properties. Additionally, the authors showed how structured approach to optical probe validation successively builds proof of probe parameters and provides several spots for go/no-go decision-making. Proof of probe parameters are critical for clinical application, and clear decision points provide efficiency and allow for early determination if a probe is worth exploring further.

Do you think IRDye dye-labeled probes could be used in your research? LI-COR Custom Services include chemistry and probe conjugation, biological assay services, translational services, and manufacturing, including cGMP manufacturing. Request a free project evaluation today.


  1. Kovar, J. L., Simpson, M. A., Geschwender, A., & Olive, D. M. (2007, August 1). A Systematic Approach to the Development of Fluorescent Contrast Agents for Optical Imaging of Mouse Cancer Models. Analytical Biochemistry, 367(1), 1-12. doi:10.1016/j.ab.2007.04.011
  2. Gong, H., Kovar, J. L., Cheung, L., Rosenthal, E. L., & Olive, D. M. (2014, February). A Comparative Study of Affibody, Panitumumab, and EGF for Near-Infrared Fluorescence Imaging of EGFR- and EGFRvIII-expressing Tumors. Cancer Biology & Therapy, 15(2), 185-193. doi:10.4161/cbt.26719

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.

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:

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!

Studying bone growth, changes, tumors? Try IRDye® BoneTag™ Optical Probes.

BrightSite Optical Imaging Agents
IRDye BoneTag optical imaging agents are tetracycline derivatives that incorporate into mineralizing bone. Structural imaging of bone can be used to more precisely localize an area of disease. A second disease-specific targeting agent with a spectrally-distinct fluorescent label can be used to localize and track disease (such as a tumor) in the same animal. When the two images are overlaid, bone structure is displayed in one color and the other target appears in a different color.

IRDye 680RD BoneTag and IRDye 800CW BoneTag are part of the ready-to-use BrightSite™ optical agents family and make it easy to begin animal studies immediately. These bright fluorescent agents are labeled with IRDye fluorophores for NIR fluorescence optical imaging, and they target a variety of disease characteristics. Simply administer the agent, then image with any small animal imaging equipment with appropriate 680 nm or 800 nm filter sets. No engineered cells or animals are needed.

IRDye BoneTag agent for imaging bone structure and remodeling

Figure 1. IRDye 680 BoneTag agent for imaging of bone structure and remodeling. Tetracycline-derived probe reveals skeletal structure, and signal is stable for weeks. Dorsal view of mouse imaged with IRDye 680 BoneTag. Image acquired with Pearl® Small Animal Imaging System.

We’ll be at AACR in Chicago, April 1 – 4, Booth 3800. Stop by and talk to us about how you can start your small animal in vivo imaging experiments today.

Try Microscopy with Near-Infrared Fluorescent Dyes For Outstanding Images

Do you know that LI-COR® near-infrared dyes and reagents can be used to perform microscopy? Absolutely! While we do not sell microscopes or offer microscopic equipment, we have evaluated the near-infrared detection capabilities of microscopes from several manufacturers, particularly in the ~800 nm wavelength region.

Here are some examples of what you can do with near-infrared dyes and reagents:

Deconvolved image of IRDye 800CW EGF binding to an A431 cell.

Figure 1. Deconvolved image of IRDye 800CW EGF binding to an A431 cell. Red represents IRDye 800CW EGF (P/N 926-08446); green represents Sytox Green nuclear stain (Invitrogen). Image captured using a Zeiss AxioImager microscope outfitted with xenon light source, IRDye 800CW custom filter set from Chroma Technology (EX: HQ760/40x, DC: 790DCXR, EM: HQ830/50m), and CCD camera with extended spectral range.

Staining of duplicated centrosomes.

Figure 2. Staining of duplicated centrosomes. Condensed chromosomes are stained with DAPI (blue). The two centrosomes (red dots) are stained with a primary antibody against pericentrin (a centrosomal component) and IRDye 800 secondary antibody. Image captured with a Leica DM RXA epifluorescent deconvolution microscope. Outfitted with xenon light source, IRDye 800 filter set from Chroma Technology (EX: HQ740/35x, DC: 770DCXR, EM: HQ780LP), and Cooke Sensicam CCD camera without extended spectral range (quantum efficiency for IRDye 800 emission ~5-10%). Images courtesy of Mark Winey and Harold Fisk, Dept. of Molecular, Cellular, and Developmental Biology, Univ of Colorado Boulder.

Learn more about what you can do with near-infrared fluorescence detection when performing microscopy.

Advantages of using PSVue® 794 for Imaging Apoptosis

PSVue® 794 is a near-infrared fluorescent probe for detection of apoptotic and necrotic cells, bacteria, and other anionic membranes. The compound exhibits fluorescence excitation maximum at 794nm and emission maximum at 810 nm and through its zinc(II)-dipicolylamine (Zn-DPA) moiety, it has been found to bind strongly to negatively charged bacterial cell walls (e.g. S. aureus, E. coli) and necrotic regions present in various tumors (e.g. mammary, prostate, glioma) in vitro and in vivo. In particular, it has also been found to bind to the phosphatidylserine (PS) residues exposed on the cell surface of apoptotic cells, making it a more cost-effective alternative to fluorescently-labeled Annexin V in various cell death assays.

Figure 1. MPTP was used to induce cell death in mouse brains as a model for Parkinson’s Disease. C57BI/6 mice were treated with MPTP to selectively destroy dopaminergic neurons. Mice were then injected with PSVue dye or control dye and imaged on the Pearl® Imager 68 hrs post injection. A. control (i.e. non-targeting) dye; B. and C. PSVue dye; D. excised brains from the three animals.

Download a scientific poster presenting information on the use of PSvue 794 in studying Alzheimer’s Disease, Parkinson’s Diesase, and contact dermatitis in mouse models.

For more near-infrared fluorescent probes, learn about BrightSite™ Small Animal Imaging Agents and CellVue® Burgundy Fluorescent and CellVue NIR Fluorescent Cell Labeling Kits.

New Tools for Cancer Surgeons: Targeted Fluorescent Imaging Probes

LI-COR interviewed Dr. Go van Dam, a surgeon specializing in oncology at the Groningen University Medical Center in the Netherlands.

A key focus of van Dam’s research is to explore new tools such as targeted fluorescent imaging probes that will help address the challenges facing oncology surgeons. He discusses his research using near-infrared fluorescent imaging during surgery to improve cancer patient outcomes. Watch an interview with Dr. van Dam.

Vasilis Ntziachristos, PhD,  Technische Universität München, Germany and Gooitzen M. van Dam, MD, PhD, University Medical Center Groningen, Netherlands presented “Shining New Light on Clinical Fluorescence Imaging” at World Molecular Congress in San Diego, CA in September 2011.