Dec. 13, 2002 Imagine if molecular and cell biologists could watch proteins and cells at work in their natural habitat in the same way that wildlife biologists observe animals in the wild. They'd sit back and witness first hand their microscopic subjects' daily routines, interactions and movements, and the places they prefer to be. This fantasy is rapidly becoming a reality thanks to new Rockefeller University research that takes advantage of a technology originally developed in the early 1980s for use in computers. Called "quantum dots," these fluorescent nanocrystals can be made to glow brightly in any desired color and thus for years have glimmered in the eyes of biologists hoping to use them for molecular and cellular imaging. But, while their potential has been clear, scientists have not been able to persuade living biological tissues to explicitly and safely take up the synthetic dots - until now.
In the Jan. issue of Nature Biotechnology, the Rockefeller University researchers demonstrate for the first time how quantum dots can be used to simultaneously track multiple living proteins or cells for up to days at a time. A fluorescent microscope is all that is required to follow the minute-by-minute activities of the color-coded proteins and cells.
"To truly understand the function of proteins and cells in their natural environment, we need to be able to watch them go about their normal business in real time," says Sanford Simon, Ph.D., head of the Laboratory of Cellular Biophysics at Rockefeller and principal investigator of the study.
Quantum dots may have medical applications as well as biological ones. For example, in such diseases as cystic fibrosis and Alzheimer's, certain proteins travel to the wrong places; like a milk truck delivering its goods to a post office instead of the grocery store, the aberrant proteins disrupt the working community of the cell. By using quantum dots to follow these proteins once they are produced in body cells, scientists can get a better handle on what goes wrong in the sorting process that leads to disease.
"Quantum dots are an incredibly powerful tool. I am sure there are many biological and medical applications we haven't thought of yet," says Jyoti Jaiswal, Ph.D., a postdoctoral associate at Rockefeller and first author of the paper.
Other authors of this study include Hedi Mattoussi and J. Matthew Mauro of the U.S. Naval Research Laboratory, Washington, D.C.
Currently, researchers visualize proteins and cells by labeling them with organic fluorescent dyes, or fluorophores, such as the popular green fluorescent protein (GFP) produced naturally by jellyfish. But this approach has several limitations.
The first has to do with how researchers induce dyes to emit light of a certain color, or spectra. For example, to make GFP produce green light, the scientists must first hit it with a laser light of a shorter wavelength, such as blue. But, if another dye were being used at the same time, one that fluoresces in the blue wavelength, then its signal would be lost in the blue light needed to trigger the first dye. Such spectral overlap limits the use of fluorophores to two, sometimes three, in any given experiment.
A second limitation of fluorophores is that they don't shine brightly for very long.
The unique physical properties of quantum dots overcome these obstacles. Simply by altering their size, scientists can manufacture them to produce light in any color of the rainbow, and, additionally, only one wavelength of light is required to illuminate all of the different-colored dots. Thus, spectral overlap no longer limits the number of colors that can be used at once in an experiment. In addition, quantum dots do not stop glowing even after being visualized for very long periods of time: compared to most known fluorescent dyes, they shine for an average of 1,000 times longer.
But while quantum dots solve these problems, they have limitations of their own - the biggest one being their water-fearing or "hydrophobic" nature. For quantum dots to mix with the watery contents of a cell, they have to possess a water-loving, or "hydrophilic" coat. Three years ago, Simon and Jaiswal's colleagues at the U.S. Naval Research Laboratory made their dots biocompatible by enveloping them in a layer of the negatively charged dihydroxylipoic acid (DHLA).
In the same study, the researchers overcame a second major obstacle of making quantum dots biologically useful - building protein-specific dots. By linking antibodies specific for an experimental protein to the DHLA-capped dots, they were able to demonstrate protein-specificity in a test tube.
In the present study, the Rockefeller scientists in collaboration with their U.S. Naval Research Laboratory colleagues have again synthesized protein-specific quantum dots, but this time they have shown their efficacy in living cells - a first for this budding technology. To do this, the researchers employed two different methods of synthesizing the quantum dots, both of which involved linking the negatively charged DHLA-capped dots to positively charged molecules - either avidin or protein G bioengineered to bear a positively charged tail. Because avidin and protein G can be made to readily bind antibodies, the researchers could then attach the dots to their protein-specific antibody of choice.
The critical test was to determine specificity: can quantum dots achieve the same exquisite selectivity that occurs when a protein is synthesized fused to GFP? To answer this question, Simon and colleagues engineered a population of cells growing together in a dish to randomly produce different levels of a membrane protein fused to GFP. When these cells were incubated with quantum dots conjugated to an antibody specific for that membrane protein, the pattern of GFP fluorescence matched the fluorescence of the quantum dots. However, the fluorescence of quantum dots lasted immeasurably longer, and the proteins could now be imaged in a rainbow of colors.
"Researchers should now be able to rapidly create an assortment of quantum dots that specifically bind to several proteins of interest," says Jaiswal.
Uncharted cellular terrain
Proteins aren't the only subjects the researchers successfully lit up with quantum dots: cells too were labeled and observed in their normal setting for very long periods of time. In the Nature Biotechnology paper, the researchers monitored human tissue culture cells tagged with quantum dots over two weeks with no adverse effects on cells. They also continuously observed slime mold cells labeled with quantum dots through 14 hours of growth and development without detecting any damage. This type of cell-tracking approach would allow researchers to study cell fate either outside the body in culture, or in whole developing organisms.
"With quantum dots, you could follow each cell in the worm C. elegans continuously from its birth in an embryo to its final destination in an adult three and a half days later," says Jaiswal.
Interestingly, the researchers discovered that they could label the cells with quantum dots using a natural process known as endocytosis, whereby cells engulf vitamins and nutrients from their outside surroundings.
"By having the cells take up the dots on their own, you reduce the risk of damaging them," says Simon.
Finally, taking advantage of their newfound ability to color-code slime mold cells, the researchers answered a long standing question about their behavior. When starved, slime molds - which typically exist as single-celled creatures - protect themselves by coming together to form one slug-shaped, multicellular organism. Scientists know that the starved cells possess the ability to instruct other nearby cells to take shape around them, while non-starved cells do not. However, they did not know the extent to which starvation differentially affects this ability.
By tagging non-starved, short-term starved and long-term starved slime mold cells with three different colors and watching their behavior under a microscope, the researchers were able to solve this riddle. It turns out that any starved cell, no matter how starved it may be, has the same ability to induce neighboring cells to undergo development.
In this experiment, quantum dots illuminated the answer to a question that heretofore lay hidden in the dark. What other biological and medical problems will yield under the power of their glow? According to Jaiswal, the possibilities are numerous. "We now have the freedom to ask all sorts of new questions."
To view animated movies of the quantum dot-labeled slime molds in action, visit the researchers Web site at: http://www.rockefeller.edu/labheads/simon/simon-lab.html.
Founded by John D. Rockefeller in 1901, The Rockefeller University was this nation's first biomedical research university. Today it is internationally renowned for research and graduate education in the biomedical sciences, chemistry, bioinformatics and physics. A total of 21 scientists associated with the university have received the Nobel Prize in medicine and physiology or chemistry, 16 Rockefeller scientists have received Lasker Awards, have been named MacArthur Fellows and 11 have garnered the National Medical of Science. More than a third of the current faculty are elected members of the National Academy of Sciences.
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