Cornell University Professor of Engineering, Applied and Engineering Physics Walt W. Webb and his group have shown (click thru to the page to see cool pictures).
ITHACA, N.Y. -- Tiny blood vessels, viewed beneath a mouse's skin with a newly developed application of multiphoton microscopy, appear so bright and vivid in high-resolution images that researchers can see the vessel walls ripple with each heartbeat -- 640 times a minute.
The capillaries are illuminated in unprecedented detail using fluorescence imaging labels, which are molecule-size nanocrystals called quantum dots circulating through the bloodstream. Quantum dots are microscopic metal or semiconductor boxes (in this case cadmium selenide-zinc sulfide) that hold a certain number of electrons and, thus, have a wide number of potential applications in electronics and photonics.
Writing in the latest issue of the journal Science (May 30, 2003), researchers at Cornell University and a nanocrystal manufacturer, Quantum Dot Corp., report that the nanocrystals are particularly useful for producing high-resolution, three-dimensional images inside living tissue.
"We have demonstrated a new approach to using quantum dots for biological studies of living animals," says Watt W. Webb, Cornell's S.B. Eckert Professor of Engineering and professor of applied physics, co-inventor of multiphoton microscopy (with Winfried Denk) and leader of the experimental imaging team at Cornell.
"Of course, there are easier ways to take a mouse's pulse," says Webb's Cornell collaborator, senior research associate Warren R. Zipfel, "but this kind of resolution and high signal-to-noise illustrates how useful multiphoton microscopy with quantum dots can become, in a biological research context, for tracking cells and visualizing tissue structures deep inside living animals."
Zipfel cited the study of vascular changes in cancer tumors as one possible application, cautioning that the Cornell researchers are not ready to recommend human-medicine clinical applications for quantum dot imaging, in part because some of the best fluorescing nanocrystals have unknown toxicity. However, mice used in the Cornell study are still alive and apparently healthy, months later, and are being monitored for long-term effects of their treatments.The Cornell researchers used quantum dots for fluorescence imaging microscopy because when excited by light, they emit bright fluorescence in different colors, according to their size, reports biophysics graduate student Daniel Larson. The quantum dots were 6 to 10 nanometers in diameter. (A nanometer is one one-billionth of a meter. By comparison, a red blood cell, at 7 millionths of a meter, is a thousand times bigger). "Even with their water-soluble coating, which is something like being encased in a soap bubble, the quantum dots are only about 24 nanometers in diameter," Larson notes.
Webb explains that the laser scanning microscope used in multiphoton microscopy is particularly adept at producing high-resolution, three-dimensional images inside living tissue because it combines the energies of two photons, striking a molecule at the same time, with an additive effect. Under the conditions used, this only occurs at the focus of the laser, so only at that point is the molecule excited to a state that results in fluorescence emission. This excitation is the same as if it arose from the absorption of a single photon of higher energy, but it is three-dimensionally localized since it is only occurring at the beam focus. The scanning microscope moves the laser beam across the area being imaged at a precise depth. When repeated scans at different planes of focus are "stacked," the result is a brightly lit and vividly detailed three-dimensional image -- and video that takes a viewer inside a living organism..
Because of the special properties of the nanoparticles, multiphoton microscopy with quantum-dot imaging can be 1,000 times brighter in tissue than conventional organic fluorophores (the chemical labels that are temporarily added to samples), says Webb. "We looked to quantum dots for even brighter images at better resolution, and that's what we found."
Results presented in the Science report show highly detailed images of capillaries beneath the skin of a living mouse after quantum dots were injected through a vein in its tail, as well as capillaries through the adipose (fat) layer around the mouse's ovaries. The researchers were particularly surprised at the saw-toothed ripples in the walls of one capillary image -- until they made a calculation. Noting the time it took to scan that part of the tiny blood vessel and the animal's heart rate during the experiment, they determined that each ripple represented the undulation of the capillary wall from one heartbeat.
Besides demonstrating the feasibility of microscopic angiography with quantum-dot labeling through skin and adipose tissue -- two of the most challenging tissue types -- the researchers said they had resolved several fundamental questions, including the fact that sometimes as many as half the dots in a preparation are not fluorescent.
Other authors of the Science article are Marcel P. Bruchez, principal scientist at Quantum Dots; Rebecca M. Williams, a research associate with the National Institutes of Health (NIH)-funded Bioimaging Resource at Cornell; Frank Wise, professor of applied and engineering physics; and Stephen W. Clark, a graduate student in Wise's laboratory. Funding came from NIH, the Defense Advanced Research Projects Agency and the National Science Foundation.
Note that as part of their quantum dot compound they used cadmium which is a toxic metal. For human imaging the development of quantum dots that have less potential for toxicity is desireable. Another problem with this approach is that since it uses light it would require the use of endoscopes to image internal organs. Still, the increased level of detail would be valuable in many circumstances.
The ability to image capillaries is of particular interest because capillary growth is a crucial element of cancer tumor growth. The ability to study this process with quantum dots (e,g, to test anti-angiogenensis compounds) will be useful for cancer research.
It is possible that quantum dots could be used in tricky ways to detect where new capillaries are growing and hence where a tumor is growing. If quantum dots could be developed that would mark existing capillaries in a way that persisted for weeks then this could ability be used in such a way that new capillary growth and hence new tumor growth could be detected. It is possible to make different types of quantum dots that emit at different frequencies. What could be done is to mark existing capillaries with quantum dots that emit light at once frequency and then a few days, weeks, or months later come back and inject quantum dots that emitted at a different frequency. Then imaging of an organ in a way that looked for each frequency of light could be done to detect capillaries that have capillaries that only emit at the frequency that the quantum dots from the second injection emit. Those capillaries that emitted at only one frequecy would be new growth capillaries and probably an indication of new growth tumor cells.
The using of quantum dots, rather than conventional dyes, resulted in a thousand-fold increase in resolution, says Webb. Additional studies found that the technique works well in fat tissue as well as through skin. "And they both scatter light like mad," he notes.
Another possible use of quantum dot imaging would be as a more sensitive method for detecting circulatory problems.
|Share |||Randall Parker, 2003 May 30 11:08 PM Nanotech for Biotech|