Qdot® Probes for In Vivo Applications

In vivo imaging using Qdot® probes

The unique photochemical properties of Qdot® probes offer an exciting and powerful tool for opening new directions in in vivo imaging applications. Whether it’s tracking the effectiveness of a disease-treating drug, elucidating fundamental processes in living organisms, or assessing the role of stem cell differentiation in vivo, applications using this novel technology in vivo are rapidly increasing, and hold far-reaching potential. Researchers performing fluorescence imaging in vivo often encounter a broad array of technical challenges, including tissue autofluorescence, fluorophore stability, and difficult quantitation, that can have a significant negative impact on data quality. The features of the Qdot® probes found in the Qtracker® non-targeted quantum dots and the Qtracker® cell labeling kits make them useful for overcoming these technical challenges to achieve meaningful in vivo data.

On this page:

• The advantages of Qdot® probes for in vivo applications
  
• Two types of Qtracker® products for different in vivo experimental approaches
  
• In vivo vascular imaging with Qtracker® non-targeted nanocrystals
  
• Multi-generation cell tracking in vivo with Qtracker® Cell Labeling Kits
  
• Overview of in vivo imaging applications
• Ordering information

The Qdot® probes used for small animal imaging provide researchers with novel attributes that help ensure their experiments are successful including:

• An exceptionally bright, stable signal for clear, extended visualization
• A choice of red-shifted emission wavelengths for increased tissue penetration
• Excitation from a single excitation wavelength for multiplexing capabilities
  

To support in vivo research, two types of products are available; the Qtracker® non-targeted quantum dots for the analysis of normal and tumor vasculature and the Qtracker® cell labeling kits to track cells for multiple generations.

Selecting a cell labeling or non-targeted Qtracker® approach

Ideal for vascular imaging, the Qtracker® non-targeted quantum dots provide a novel in vivo solution for viewing normal vasculature down to the capillaries. These non-targeted quantum dots are specially designed with a modified surface for reduced non-specific binding and immune response. That makes these useful for in vivo imaging and the imaging of micro-injected cells. In contrast to other vasculature agents, the Qdot® probes have minimal leakage from the vasculature, and allow imaging for at least 3 hours. Learn more about in vivo experiments imaging vasculature.

It should be noted that these materials have been specifically developed to reduce interactions with other molecules, and are therefore not suitable for conjugation. Conjugatable Qdot® probes in several colors are available as ITK carboxyl or amino-PEG quantum dots or in the Qdot® Antibody Conjugation Kit.

The Qtracker® Cell Labeling Kits provide reagents to deliver Qdot® probes into live cells. One potential in vivo application is to label stem cells using the Qtracker® Cell Labeling Kit prior to introduction into animals. The position and migration of the stem cells can be tracked for up to at least 5 divisions via non-invasive or minimally invasive approaches. Alternatively, tissue analysis of sacrificed animals can be performed. Because the Qtracker® labels do not leak out of the cells, they will not be taken up by other cells in the animal model (Figure 1).

Figure 1. U-118 3T3 and HeLa cells labeled with Qtracker® labels demonstrate high cellular retention.

Sentinel lymph node mapping

In "Near-Infrared Fluorescent Type-II Quantum Dots for Sentinel Lymph Node Mapping," (Kim et al. Nature Biotech. 2004, 22:93-97) researchers at MIT and Massachusetts General Hospital describe the development of an improved method for performing sentinel lymph node biopsy, a crucial first step in determining whether a cancer has spread to other parts of the body. The new method depends on the use of near-infrared–emitting quantum dots to illuminate lymph nodes to guide cancer surgery. The near-infrared quantum dots were developed and synthesized at the Massachusetts Institute of Technology's department of chemistry, in the laboratory of Professor Moungi Bawendi. The novel intra-operative, near-infrared–fluorescence imaging system was developed in the laboratory of Dr. John Frangioni, Assistant Professor of Medicine and Radiology at Harvard Medical School and an Attending Physician at Beth Israel Deaconess Medical Center. Roger F. Uren of the University of Sydney provides a perspective on this work in Nature Biotech. 22:38-39.

Sentinel lymph node (SLN) mapping is a common procedure used to identify the presence of cancer in a single, "sentinel" lymph node, thus avoiding the removal of a patient's entire lymph system. SLN mapping currently relies on a combination of radioactivity and organic dyes but the technique is inexact during surgery, often leading to removal of much more of the lymph system than necessary, causing unwanted trauma. The current work was performed on laboratory animals, including pigs, considered by scientists to be a good predictor of human results.

The authors first injected near-infrared–fluorescent quantum dots (peak emission 840-860 nm in neutral aqueous buffer) intra-dermally into the paw of a mouse; the quantum dots entered the lymphatic system and migrated to an axillary location. Re-injection and colocalization of isosulfan blue with the fluorescence signal confirmed the site to be the SLN. The authors injected 400 pmol of near-infrared quantum dots intra-dermally into five pigs and followed them visually to the lymph system 1 cm beneath the skin of the animals. Localization of the SLN required only 3–4 minutes. The new imaging technique allowed the surgeons to see the target lymph nodes clearly with no invasive surgery.

The study reported that the imaging system with near-infrared quantum dots was a significant improvement over the dye/radioactivity method currently used to perform SLN for several reasons, including: throughout the procedure, the quantum dots were clearly visible using the imaging system, allowing the surgeon to see not only the lymph nodes, but also the underlying anatomy; the imaging system and quantum dots allowed the pathologist to focus on specific parts of the SLN that would be most likely to contain malignant cells, if cancer were present; and the imaging system and quantum dots minimized inaccuracies and permitted real-time confirmation of the total removal of the target lymph nodes, drastically reducing the potential for repeated procedures.

The near-infrared region of the spectrum promises to be increasingly important in bio-medical applications, particularly in vivo, as near-infrared–emitting quantum dots become more widely available. Tissues and blood absorb very little light in this region and autofluorescence is also minimal. In addition, light scattering is a strong function of the wavelength of the excitation and emission wavelength; dyes that emit in the near-infrared are capable of being imaged with relatively little loss of signal and resolution compared with visible or UV dyes. It is exceptionally difficult to prepare stable near-infrared dyes using traditional organic approaches, and until recently this spectral region has not been adequately exploited.

It is actually quite difficult even to prepare reasonably stable quantum dots for use in the near-infrared.Type II quantum dots such as those used by Professor Bawendi's group depend upon two different semiconductor materials which are blended in the correct proportions and geometries to result in emission at a much lower energy than is accessible by either of the materials alone.

Integrin-positive tumor targeting

In a published report from Cai et. al. (Nano Letters. 2006, vol.6 no.4 p669-676) a group of researchers from Stanford University demonstrate the use of arginine-glycine-aspartic (RGD) peptide-labeled quantum dots to target integrin-positive tumor vasculature in a mouse xenograft model. Integrin binds to RGD containing components of the interstitial matrix, and is an important component in tumor angiogenesis and metastasis. Using non-invasive imaging, quantum dot-RGD probes in the the near infrared (NIR) fluorescence targeted to integrin were visualized.

Once the quantum dot 705-RGD conjugate was made and demonstrated to bind to integrin in multiple cell lines, but not to integrin-negative cells, this was used in ex vivo studies to further demonstrate the specificity of binding. Cai et.al. show that this probe specifically recognized the tumor in animals. The quantum dot 705-RGD and control quantum dot 705 were introduced into tumor bearing mice. Using the Maestro™ from Cambridge Research Instrument (CRi) company, in vivo results showed the increase in tumor fluorescence intensity. Tumors from sacrificed mice were harvested and imaged using the IVIS 200™ System from Xenogen show the presence of the quantum dot 705-RGD probe.

This paper provides data that suggests a potential for using quantum dots as a universal approach to detect many tumor types, and for an ultimate use for optical imaging in cancer diagnosis and imaging-guided surgery.

Single-quantum dot tracking of receptor dynamics

Researchers at INSERM (Paris) and CNRS (Paris) have utilized the incomparable brightness and photostability displayed by Qdot® probes to track the motional dynamics of neuronal glycine receptors (Dahan, M.; Lévi, S.; Luccardini, C.; Rostaing, P.; Riveau, B.; Triller, A. "Diffusion Dynamics of Glycine Receptors Revealed by Single-Quantum Dot Tracking," Science, 2003, 302, 442-445). The ability to image the quantum dot labels not only by real-time fluorescence, but also by high-resolution electron microscopy allowed both dynamic behavior and accurate localization studies to be performed with the same samples.

Receptor dynamics in the synaptic cleft have eluded analysis because the size of the cleft precludes use of large probes, such as 40-nm gold particles or 500-nm latex beads. Furthermore, real-time visualization of receptor mobility using small fluorescent labels has been limited by photobleaching. Dahan et al. studied the mobility of glycine receptors (GlyR) in living neurons with an aim to developing a new approach that would allow access to the synapse and could be tracked for longer periods of time. The investigators studied GlyR1 subunits at the surface of spinal cultured neurons using a primary antibody, biotinylated anti-mouse Fab fragments and Qdot 605 streptavidin conjugates. GlyRs were detected within synaptic and extrasynaptic domains.

Trajectories of single Qdot® probe–GlyR complexes in the membrane could be visualized for at least 20 minutes, compared to ~5 seconds for Cy®3 dye conjugates. The signal-to-noise ratio for the Qdot® conjugates was almost an order of magnitude greater than was observed for Cy®3 dye, allowing a resolution of 5–10 nm versus 40 nm with Cy®3 dye. Single Qdot probe tracking of rapid lateral dynamics of GlyRs enabled observation of multiple exchanges between extrasynaptic and synaptic domains. The high photostability of Qdot® probes allowed the tracking of individual GlyRs in the same neuritic region using time lapse recording of one 75-ms image per second for 20 minutes. Observed patterns of diffusion led the investigators to classify receptors as synaptic, perisynaptic or extrasynaptic. Furthermore, the Qdot conjugates did not appear to alter the estimates of diffusion coefficient values because the investigators recorded comparable proportions of rapidly diffusing receptors with Cy®3 dye and Qdot® probes.

Electron microscopic images of silver-intensified Qdot® probes showed that nanocrystal–GlyR complexes could access the core of the synapse. Qdot® probes were never detected intracellularly and thus do not appear to be internalized. The authors concluded that Qdot® probes offer a unique tool for acquiring both fluorescence and electron microscopy images with the same probes, providing access to two types of information: temporal dynamics and high-resolution cellular localization of single biological molecules.

In vivo fluorescence imaging

Researchers at Cornell University have been exploring in vivo fluorescence imaging using Qdot nanocrystals (Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. "Water Soluble Quantum Dots for Multiphoton Fluorescence Imaging In Vivo," Science 2003, 300, 1434-1436). Images were acquired following tail vein injections of 550 nm-emitting Qdot® probes which had been solubilized in buffer using patented and proprietary technologies. Fluorescently labeled vasculature was visible through skin and adipose tissues, which have both proven to be problematic in the past due to excessive scatter. Attempted visualization using FITC-dextran was much less successful.

The Cornell team, comprising internationally recognized experts in the field of multiphoton spectroscopy, found that Qdot nanocrystal materials are characterized by two-photon absorbance cross sections of up to 47,000 Goeppert–Mayer units, "by far the largest of any label used in multiphoton microscopy." In other words, under two-photon excitation conditions, Qdot® probes absorb a great deal of the incident light, which makes them exceptionally bright compared to conventional dyes.

Two-photon techniques promise to revolutionize deep tissue imaging. Biological tissues are turbid media that scatter light very intensely. This scattering usually filters excitation light so that very little can usually be transmitted more than a few microns through tissues. Scattering efficiency is a strong function of excitation wavelength, however, with blue being more problematic than red. In two-photon experiments, red or infrared excitation light is highly focused at a tunable depth. In the unfocused region above and below the target depth, the light is too weak to be absorbed; as a result, only a very narrow depth of field is imaged, as in more familiar confocal techniques. By changing the focal depth, slices can be acquired and combined to build up a three-dimensional view of the tissue.

Immunofluorescent labeling of breast cancer markers

In one publication (Wu et al. "Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots" (2003) Nature Biotechnology 21: 41-46), scientists described the use of nanocrystals coated with an amphiphilic polymer linked to immunoglobulin G or streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells. In addition, these nanocrystal conjugates were used to stain actin and microtubule fibers in the cytoplasm and to detect nuclear antigens inside the nucleus. Wu et al. observed that all labeling signals were specific for the intended target. The specific signals obtained using the nanocrystal conjugates were brighter and considerably more photostable than comparable organic dyes in the same experiments. Wu et al. also conducted a multiplex experiment to visualize two targets simultaneously using nanocrystals with different emission maxima (535 nm and 630 nm) coupled to the same ligand (streptavidin) or to different ligands (IgG and streptavidin).

Although the use of quantum dot conjugates to carry out multicolor labeling of cells and tissues is becoming increasingly common, this work specifically addressed performance issues such as photostability. Their results showed that little if any photodegradation of the nanocrystal signal can be seen under conditions in which the organic dye is almost completely photobleached. To ensure this was a result of the inherent properties of the nanocrystals and not a function of the structures being labeled, Wu et al. additionally describe the inverse labeling experiment, which yielded similar results.

Bead-based multiplexed SNP analysis

Qdot® probe–encoded microspheres have been used for the first time in the multiplexed genotyping of nearly a hundred genomic samples (Xu et al. 2003 "Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay," Nucleic Acids Res. 31(8):e43). Polystyrene microspheres were dyed with two green colors of Qdot® probe in varying proportions to create uniquely encoded beads. Oligonucleotide probes were attached to the beads such that the composite emission spectra from each specific batch of beads could be used to identify the attached oligo spectroscopically. Two channels on a conventional flow cytometer were used to evaluate the codes.

Following multiplexed PCR amplification, the amplicons were evaluated against ten SNP types in parallel using an allele specific hybridization strategy. The results were nearly 100% accurate, even though the SNPs investigated were chosen from the highly problematic P450 family. Furthermore, since the multiplexed assays could be carried out in an extremely small volume in a single well, only 1 nanogram of DNA was required per patient.