Biologists have long been eager to probe living cells in full colour over extended periods of time. Such a technique could reveal the complex processes that take place in all living organisms in unprecedented detail, such as the development of embryos.

Existing imaging techniques use natural molecules that fluoresce, such as organic dyes and proteins that are found in jellyfish and fireflies. However, each dye emits light over a wide range of wavelengths, which means that their spectra overlap. This makes it difficult to use more than three dyes at a time in order to tag and image different biological molecules simultaneously. The fluorescence of dyes also tends to fade away quickly over time.

Inorganic semiconductor nanocrystals - quantum dots - can get round these problems. As well as being brighter and living longer than organic fluorophores, quantum dots have a broader excitation spectrum. This means that a mixture of quantum dots of different sizes can be excited by a light source with a single wavelength, allowing simultaneous detection and imaging in colour. Turning these ideas from physics into biology, however, has remained a challenge because quantum dots cannot survive in water, and they must remain non-toxic.

Now a team of physicists and biologists led by Albert Libchaber and Ali Brivanlou at Rockefeller University in the US has produced new, biocompatible quantum dots and used them to image a live frog embryo (B Dubertret et al. 2002 Science 298 1759-1762). The researchers were able to "dress" quantum dots in an organic disguise that prevents them from coming into direct contact with the aqueous biological environment.

Molecular beacons

Quantum dots are nanometre-scale crystals that were developed in the mid-1980s for optoelectronic applications. They are composed of hundreds to thousands of atoms of an inorganic semiconductor material in which electron-hole pairs can be created and confined. The size of quantum dots can be tuned with nanometre precision during chemical synthesis, which gives them intriguing optical properties.

When the electron-hole pairs in the core of a quantum dot are excited with a beam of light, they re-emit light (fluoresce) with a narrow and symmetric emission spectrum that depends directly on the size of the crystal. This means that quantum dots can be fine-tuned to emit light at a variety of wavelengths simply by altering the size of the core, and therefore constitute a set of multicoloured molecular beacons for use in imaging. A 3 nm particle made from cadmium selenide, for example, radiates green light at 520 nm, while a slightly larger 5.5 nm particle of the same material radiates red light at 630 nm.

The core of the quantum dot is usually contained within a protective inorganic shell such as zinc sulphite, which has a higher electronic band gap than the core. This improves the confinement of the electron-hole pairs and therefore increases the intensity of the fluorescence. However, it is the hydrophobic nature of this outer shell that prevents quantum dots from being used in aqueous biological environments. In addition, to ensure that they remain non-toxic and that they recognize specific targets in a cellular context, quantum dots need to be modified with biological molecules to make them biocompatible before they can be let loose in a living organism.

Watertight

Since the mid-1990s a great deal of effort has gone into the development of robust, versatile and biocompatible surface chemistries to produce nanocrystals that are both soluble and functional. Various modifications of the outer surfaces of quantum dots have been successful, but at the expense of increased size and a compromise in the fluorescent and colloidal properties of the nanocrystals.

Now the Rockefeller team has produced soluble nanocrystals by a rapid and rather simple one-step procedure that does not require any surface modification of the particles. The breakthrough came in Albert Libchaber's lab when Vincent Noireaux and Benoit Dubertret realized that an individual hydrophobic quantum dot could be encapsulated in a micelle - a simple chemical aggregate that has a hydrophobic centre surrounded by a hydrophilic shell.

The micelle was made from two kinds of phospholipids - fat-related products that are the major constituent of cell membranes in living organisms. Natural phospholipids were mixed together with polymer-grafted phospholipids. The resulting mixture forms a micelle because the hydrophobic tails of the mixture move away from water, while the polar heads immerse themselves in aqueous environments. Quantum dots are quickly engulfed during this process and stay protected at the centre of the micelles. Aqueous suspensions that consist of cadmium-selenide quantum dots coated with zinc sulphite are stable for months within the micelles.

The crucial question, then, was whether the encapsulated quantum dots retained their optical and colloidal properties? The Rockefeller group used high-resolution transmission electron microscopy to obtain images suggesting that the majority of the micelles contained a single quantum dot. These appeared to be spherical and of the same size, and they did not form any aggregates. The fluorescence quantum yield - the ratio of the amount of light emitted from a sample to that absorbed by the sample - of the quantum-dot micelles in water was 24%, a value very similar to the original quantum dots.

Having successfully kept the quantum dots away from water, the team went on to chemically alter the surface of the micelle to allow it to act as glue onto which biological macromolecules could be attached. These modified micelles were then linked to short single-strand fragments of DNA. The highly specific nature of DNA bonding meant that the DNA-micelle could recognize and pair with its complementary DNA strand. These experiments therefore demonstrated how the quantum-dot micelles could be adapted to locate a specific target, at least in vitro.

Frogs first

The success of these experiments prompted the Rockefeller team to investigate quantum-dot micelles in a live biological system. They injected a billion of the particles into Xenopus embryos (a South African frog) that were in early stages of development, and performed in vivo imaging using time-lapse microscopy.

The researchers made several key observations. Upon cell division, the fluorescent particles appeared to be solely distributed to the offspring of the injected parent cell, and did not diffuse out of the cell. The path taken by a fluorescent cell could therefore be traced back to the cell that was first injected. The researchers also found that all embryonic cell types can be stained, which came as a pleasant surprise since the behaviour of the particles in a cellular context was totally unknown. Crucially, they found that the quantum-dot micelles do not appear to have any detrimental effect on the frog's development, and that the quantum-dot micelles were stable in vivo. The dots were still producing a detectable fluorescence signal after four days of embryonic development, and fluorescence was visible even in high-background regions such as the embryo gut. Finally, the quantum-dot micelles were found to be more resistant to photobleaching than other fluorophores (see figure 1).

The Rockefeller results clearly show that fluorescence imaging with micelle-encapsulated quantum dots can be successfully applied to a live organism. But there are still issues that have to be addressed. The team used 4 nm (green) particles only, and we need to know if quantum dots of other sizes can sustain similar micelle encapsulation. The optical properties of the particles that were observed in vitro also need to be validated experimentally in a biological system. For example, can they be excited and detected simultaneously to form a multicolour image of live cells? While a very large number of quantum dots were used to image these embryos, it would also be interesting to know the lower detection limit of the nanocrystals within a cell.

Furthermore, although the targeting of quantum dots to specific biological objects has been demonstrated in principle using DNA, this has not yet been verified in a living biological system. It remains to be seen if the surface of the micelle can be used to target macromolecules or ligands of interest, in order to penetrate cells and pinpoint their constituents.

But quantum-dot imaging is itself at an embryonic stage. After fascinating physicists for over a decade, fluorescent semiconductor nanocrystals are finally fulfilling their promise in the biology lab. Fluorescence-based techniques are already widely used in biology, so we can expect that quantum dots will rapidly expand the biologist's toolbox.