Quantum dots push the boundaries of fluorescent tagging for microscopic analysis of cellular processes.

This image contains multicolor immunofluorescence imaging with Invitrogen’s Qdot secondary antibody conjugates of a mouse intestine section.  (Source: Thomas Deerinck and Mark Ellisman, The National Center for Microscopy and Imaging Research)

How large is a dot?

This sounds like a question some ancient philosopher would have asked his students. But nanoparticle scientists may have answered this question by creating a technology known as the quantum dot. Colloid-based semiconductors by nature, quantum dots are used in computing applications, photovoltaic devices, light-emitting devices, and, since the year 2000, have seen increasing use as a fluorescence-based label for microscopic analysis of cells. And, according to its proponents, this labeling method has its advantages.

“With quantum dots, you can see single-molecule interactions,” says Martin Hetzer, PhD, an assistant professor at the Salk Institute for Biological Sciences, La Jolla, Calif. “The advantage there is that you can be extremely sensitive because you can see a single quantum dot.” Hetzer uses quantum dots for both a cell-free, in vitro application and for live-cell imaging. For the in vitro analysis, Hetzer uses quantum dots to monitor biomolecular interactions. In this scenario, one isolated protein in the interaction is immobilized on a single, affinity chromatography bead and a mixture of eight different isolated proteins is labeled with quantum dots; each protein is labeled with a different colored quantum dot. Then, the presence of an interaction is determined by measuring the intensity of fluorescence on the surface of the bead.

From many to one
According to Hetzer, the use of quantum dots differs from the previous method for studying these interactions where millions of beads were needed because the sensitivity was not high enough to detect the interaction. In other words, quantum dots significantly reduce the amount of material needed for the experiment. “You can actually do up to eight experiments by labeling different components with different colors,” says Hetzer.

The specific proteins Hetzer studies are those that make up the nuclear pore complex—a very large, multi-protein assembly that controls molecular transport between the nucleus and cytoplasm of the cell in eukaryotes. “The really important part is that many of these proteins are really large and hard to express. And if you can express them, you can only express a very limited amount of protein. But our assay is sensitive enough so you can actually work with very little protein and still get the same result that you would get if you had a highly-expressed, recombinant protein,” says Hetzer.

click to enlarge   Epifluorescence images of wild type Hen Egg White Lysozyme (HEWL) conjugated to CdSe/ZnS quantum dots (QDs) (A) and HEWL amyloids conjugated to CdSe/ZnS QDs (B). (Source: Roger LeBlanc, PhD)

Hetzer is also developing a live-cell imaging protocol that uses quantum dots. “The use of quantum dots in live-cell imaging is very limited right now,” he says. “The huge bottleneck being that no one has a way to deliver quantum dots efficiently into cells. They work if you stain extracellular proteins on the outside of cells, but if you want to look at intracellular processes, then the application of quantum dots is really limited in live cells.” But he is working on this problem by developing new technologies to deliver quantum dots, thereby labeling cellular proteins in vivo.

Responding to the environment
One researcher using quantum dots to perform cellular imaging is Jay Nadeau, PhD, assistant professor of biomedical engineering at McGill University, Montréal, Quebec, Canada. Nadeau is interested in developing new fluorescent probes to study intracellular processes including action potential generation, calcium balance, and redox reactions. “And quantum dots are good for any or all of these applications because they have the potential to respond to their environment by changing their fluorescence properties,” says Nadeau. Specifically, she is working on conjugating quantum dots to a specific voltage-gated sodium channel found in the bacterium Bacillus halodurans called NaChBac in order to make this protein potentially sensitive to changes in voltage.

Nadeau is also investigating some of the different photo-physical properties of quantum dots. “So the fluorescence [emitted by quantum dots] includes what we’d call the steady-state fluorescence (or just the color you get out), but it also includes things like blinking or intermittency,” says Nadeau. She is looking at changes in photo-enhancement or photobleaching of quantum dots in response to environment. “We are trying to use these [properties] as sensing mechanisms as well as just looking at the fluorescence intensity. That’s why we are using quantum dots and not some other fluorescence-based tagging methods.”

“I have done some work to compare the work of the fluorophore with the quantum dot and there is no comparison at all. And for these three reasons: intensity of luminescence, size distribution of the particles allows for different emission spectra, and each quantum dot has a very narrow bandwidth compared to the organic fluorophore. And that is very important,” says Roger Leblanc, PhD, professor of chemistry, University of Miami, Coral Gables, Fla., who uses quantum dots for biosensing and cell imaging applications.
 
Other challenges
According to Nadeau, the biggest challenge faced when using quantum dots is inconsistency in results produced from one batch to the next. She adds that this is true whether the dots are purchased from a vendor or produced in-house. “You have to be very careful about getting the same color. If you don’t have the same color, they can be seen differently,” says Nadeau. This inconsistency also occurs as quantum dots age, causing variable results from one week to the next, even with the same batch of particles. She has been trying to improve their reproducibility by using capping agents and by repeating experiments with quantum dots of different colors.

Ben Van Houten, PhD, Chief, Program Analysis Branch & Senior Investigator in the Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, N.C., uses quantum dots to study DNA repair mechanisms in Escherichia coli. By labeling DNA repair proteins with quantum dots (QDots from Invitrogen Corporation, Carlsbad, Calif.) he is able to visualize the movement of these proteins as they “walk along the DNA towards a lesion.” Some of the approaches he uses include the “antibody sandwich” where an epitope tag was attached to one of the repair proteins, then reacted with a protein-specific primary antibody, followed by an anti-isotype secondary antibody conjugated to a quantum dot. “And this protein still worked with the big quantum dot hanging off of it,” says Van Houten.

A major challenge Van Houten faced when he started to use quantum dots was how to stoichiometrically attach the particles to proteins, i.e. one quantum dot per protein. “As you can imagine because the quantum dot has a pretty big surface area, when it is modified with bioreactive molecules, there are several different sites on the quantum dot that can react. So if your stoichiometry is wrong, you can have two target proteins bound to one quantum dot. And that would be pretty bad for visualizing single molecules,” says Van Houten. So a postdoctoral fellow in Van Houten’s lab, Hong Wang, used her expertise in atomic force microscopy to solve this issue, showing the one-to-one ratio under the right conditions. Van Houten describes the atomic force microscopy process as follows. A 50-nm tip is dragged on a silica surface covered with proteins or DNA; each protein or DNA represents the molecular equivalent of a “speed bump.” And when the tip reaches one of these bumps, it deflects a cantilever, which in turn deflects a laser, which in turn deflects a photomultiplier, thus generating a signal. “So it is a great achievement in my opinion because we were able to get the stoichiometry and we can then assess in a complex of different proteins which one is actually there.”

So whether quantum dots are used for in vitro studies of in vivo studies, the evidence is clear: they are powerful tools to label biomolecules prior to studying their activity.

This article was published in Drug Discovery & Development magazine: Vol. 11, No. 7, July, 2008, pp. 20-22.