A quantum leap for in vivo imaging

Quantum dot technology is an exciting development in the field of nanotechnology

Nanotechnology - the ability to control atoms and molecules individually and to employ them in ever more precise ways - has become a subject of much interest in recent years. The area of nanotechnology at the most advanced stage of understanding is that of nanomaterials1 and this article explores an exciting development in this field - quantum dot technology. Quantum dots (QD’s) are currently revolutionising the fluorescent imaging of cells and molecules, especially for in vivo applications.

QD’s are new inorganic probes that are allowing researchers to get clearer images of cellular processes that until now have been viewed for shorter periods and less brightly through the use of organic dyes.

Semiconductors, with a difference
QD’s consist of a nanometer sized semiconductor core, often CdSe (cadmium selenide), of 3-6nm in diameter depending on the application. It is the composition and size of QD’s that give them their extraordinary optical properties.

Larger semiconductors contain electrons that exhibit a range of energy levels. These different energy levels are so close together that they can be classed as continuous. In between these energy levels a band gap occurs; below the gap electrons reside in the valence band, above it they form the conduction band. To trigger the electrical conductivity of a semiconductor, an external stimulus is applied to move electrons from the valence and into the conduction band.

When an electron moves into the conduction band, it leaves a ‘hole’ of positive charge in the valence band. The hole-electron pair is known as an ‘exciton’. The distance between the hole and electron of an exciton is described as the Exciton Bohr Radius – a fixed value for any given semiconductor material.

In semiconductors that have been reduced to the size of QD nanocrystals, the particle size itself is smaller than the ‘natural’ Exciton Bohr Radius for that particular material. This prevents the Bohr Radius distance extending to the length it would be in a larger semiconductor molecule. Under these conditions, electron energy levels can no longer be considered as continuous but discrete - this is known as quantum confinement.

Quantum confinement
This concept is the reason why QD’s are so important. Electrons in larger semiconductors will fall back into the valance band very quickly, returning to their natural valence energy. This process causes the emission of energy as the electron crosses the band gap. Whereas QD’s are capable of confining electrons in three dimensions, in which the electrons occupy discrete energy states.

For a given input energy a QD will only emit specific spectra of light. With decreasing QD diameter, there will be a corresponding increase in energy of emitted light. For example, if a particular semiconductor absorbs light at the band gap energy of 900nm, when that semiconductor is shrunk down to a QD size it will absorb light at a smaller wavelength and also emit light at shorter wavelengths. As emission frequency depends on the band gap distance, and in QD’s this can be controlled by adjusting the dot size, it is therefore possible to have control over the emission.

Quantum dot structure
Quantum confinement occurs in the QD core. Cores are composed of cadmium sulphide (CdS), cadmium selenide (CdSe), or cadmium telluride (CdTe). The semiconductor material used for the core is chosen based upon the emission wavelength range being targeted: CdS for UV-blue, CdSe for most of the visible spectrum, CdTe for the far red and near-infrared.             

The use of QD’s in biological applications has developed rapidly since  improvements in the shell and coatings have been made. Shells isolate the core, enhance the brightness of the materials, and increase the emission stability in buffers. Emission is typically weak and unstable with unadorned cores so a non-emissive and transparent shell is used, typically zinc sulphide (ZnS) (Figure 1).   

                              
                                                              
                                                              Figure 1.                                

QD coatings differ considerably, but a unique approach by one manufacturer, the Quantum Dot Corporation, employs an outer layer of a mixed hydrophobic/hydrophilic
polymer with carboxylic acid derivatisation. The hydrophobic part of the polymer interacts with the inner coating while the hydrophilic portion interacts with the external solvent to provide solubility in buffers. For these Qdot QD’s, this has enabled unprecedented stability in nearly the full range of environments employed in the biological sciences. This novel coating also provides a flexible carboxylate surface to which many biological and non-biological moieties can be attached. The resulting surface is derivatisable with antibodies, streptavidin, lectins, nucleic acids and other molecules.

Advantages over conventional imaging methods
QD’s produce a bright and non-photobleaching emission with narrow symmetrical emission spectra. They also allow multiple colours to be excited simultaneously using a single excitation wavelength. As explained above, the colour of QD’s, both in absorption and emission, can be ‘tuned’ to any chosen wavelength by simply changing their size. Therefore by using only a small number of semiconductor materials and an array of different sizes, researchers have access to QD’s with colours that span from ultraviolet to infrared. While choice of material dictates the region of the spectrum available for emission from these particles, it is the size of the particles that tunes the emission wavelength (Figure 2).

Figure 2. Emission spectra, showing narrow, symemetric peaks of varous nanocrystals

Using conventional methods such as green fluorescent protein or dyes like rhodamine, researchers could view only a few colours at once. Each of the fluorophores must be excited with a specific wavelength of light, which can block the emitted colour of other fluorophores in the experiment. QD’s allow the simultaneous fluorescence of many different, specific colours and therefore enable the easy discrimination between tagged targets, even in a single cell.

Qdot QD’s provide many other advantages over conventional labelling, including a large extinction coefficient and high quantum yield. Excellent photostability means that whilst conventional fluorophores can fade in a couple of hours, QD’s remain stable for days or even months. Long-term cellular retention makes QD’s suitable for studying cell motility, migration, differentiation, morphology and other cellular function. In contrast to organic fluorophores, Qtracker QD’s do not leak out to be taken up by adjacent cells in the population.

The coating of Qdot QD’s protects cells from cadmium toxicity and makes it possible to attach a variety of targeting molecules. The small size of the Qdot QD’s allows them to be used as molecule-specific markers that do not interfere with normal cell processes, allowing tagged cells to grow, signal and process as usual.

In vivo applications
QD’s are also proving to be better than commonly used radiolabelling techniques for in vivo applications. This is demonstrated in the following example.

Sentinel lymph node identification and removal using quantum dots3
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.

Researchers at MIT and Massachusetts General Hospital have developed an improved method for performing SLN biopsy. This new method uses near-infrared (NIR)-emitting QD’s to illuminate lymph nodes to guide cancer surgery.

The authors first injected NIR QD’s (peak emission 840-860nm in neutral aqueous buffer) intra-dermally into a mouse, which entered the lymphatics and migrated to an axillary location. Re-injection and co-localisation of isosulfan blue with the NIR signal confirmed the site to be the SLN. The authors injected 400pmol NIR QD’s intra-dermally into five pigs and followed them visually to the lymph system 1cm beneath the skin. Localisation 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 (Figure 3).

Figure 3: Localisation of a pig sentinel lymph node through intradermal injection of near-infrared-emitting quantum dots. Top left: visible light colour image.
Top right: as viewed through a NIR-sensitive camera. Bottom left: colour and NIR images merged
 






The study reported that the imaging system with NIR QD’s was a significant improvement over the dye/radioactivity method for several reasons, including: throughout the procedure QD’s 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 QD’s 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 QD’s minimised inaccuracies and permitted real-time confirmation of the total removal of the target lymph nodes, drastically reducing the potential for repeated procedures.

Non-targeted QD’s
The following example of the use of QD’s in another in vivo application demonstrates their ability to provide excellent fluorescent cellular images. Qtracker non-targeted QD’s (Quantum Dot Corporation) are coated with polyethylene glycol (PEG) to greatly reduce non-specific binding and immune response. To increase time in circulation in vivo and reduce toxicity responses, these QD’s do not contain reactive functional groups. These materials have been specifically developed to reduce interactions with other molecules and are not designed for conjugation.

Figure 4 shows a chick embryo that was injected through the major vitelline vein with Qtracker 705 non-targeted QD’s. After a few minutes of circulation, fluorescence images of the embryo were captured at increasing magnification using 460nm excitation lamps and detecting fluorescence through an appropriate emission filter. As the images show, these reagents revealed highly detailed vascular structure at all levels of magnification (Data courtesy of Greg Fisher, Carnegie Mellon University).

Figure 4.Chick embryo injected with Qtracker 705 non-targetted QDs 

Conclusion
There are now many QD products available that enable researchers to benefit from QD technology in a wide range of applications. For example, the Quantum Dot Corporation has developed a number of kits including: Qtracker kits to deliver QD’s into the cytoplasm of live cells using a custom peptide; QD conjugates, such as the Qdot 705 Streptavidin Conjugate for utilising quantum dots in the NIR region of the spectrum; and also several antibody conjugates for use in multicolour immunofluorescence research.

QD nanocrystals possess unique properties that distinguish them from traditional fluorescent dyes. These properties can combine to produce much more intense and sensitive signals than are generated from organic dyes. With the right chemistry, QD’s give researchers the opportunity to observe labelled cells using extensive continuous illumination, without the cytotoxicity, photobleaching and degradation problems commonly associated with the use of organic fluorescent dyes.

By Helen Baker, Technical Product Manager, Cambridge BioScience, Cambridge