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  • Ultrafast Lasers Capture Brain Images
Jul 2003
SAN DIEGO, Calif., July 2 -- A new technique using ultrafast lasers to slice and take images of brain tissue has been developed by an interdisciplinary team of scientists headed by physicists at the University of California, San Diego (UCSD). The technique, described in the July 3 issue of the journal Neuron, provides a new way to automate and modernize histology -- the study of tissues at the microscopic level -- and to map the production of neurotransmitters and other proteins involved in communication between cells and normal cell function, which are produced in different regions of the brain.

NO MORE BRAIN FREEZE: 3-D image of mouse brain showing blood vessels using laser technique. Photo: Philbert Tsai, University of California, San Diego

"We can now look at a large portion of the brain and do statistics on the anatomy, because this new technique is able to more precisely measure structures in the brain and to do it without the tedious and laborious methods of thin-slicing sections of the brain," said David Kleinfeld, a professor of physics at UCSD, who headed the study. "It solves a big problem and it potentially changes the way a lot of science is done today."

Until now, to get microscopic brain images, scientists have relied on a procedure developed in the early twentieth century that involves manually cutting thin slices of frozen brain and viewing them through a light microscope. Not only is this painstaking, but freezing can damage the tissue.

In the new technique developed by the Kleinfeld team, the tissue is imaged and ablated, or vaporized, in successive iterations. The ablation requires a femtosecond laser, which produces light pulses lasting one quadrillionth of a second. This is as short in comparison with a second as a second is in comparison with all of human history.

"The pulse is so short that it doesn't generate heat, which would damage the tissue," according to Philbert Tsai, a graduate student in Kleinfeld's lab and the first author of the study. "Electrons are ripped away from atoms to form a plasma on a time scale too short for the atom cores to vibrate and produce heat."

The layer of tissue removed in each successive laser ablation is narrower than a human hair. After each ablation, the newly uncovered tissue is labeled with a fluorescent dye, and lower intensity laser light is used to take an image of the surface. Successive snapshots of each layer can be recombined to create a 3-D image. Iit takes about a day to image the entire brain of a mouse, but the procedure can be fully automated.

One particularly valuable application of this technique is to image brains of animals that have been genetically engineered to produce a fluorescent protein in certain cells. By studying 3-D images of the brains of mice that produced fluorescent protein in the blood vessels of the brain, Kleinfeld's team was able to determine the volume of the blood vessels relative to the rest of the brain. Images of brains that express a particular fluorescent protein could also be used to quantify how much of that protein there is in different regions of the brain. This technique is not limited to the brain, either -- other tissues in the body could be imaged with the femtosecond laser. The only disadvantage is that the sample is destroyed in the process, so it would not be suitable if, for example, a clinician wanted to preserve a sample for later reference.

The project drew together researchers with a wide range of expertise. Jeffrey Squier, a physicist and laser expert in the chemistry department at UCSD and now at the Colorado School of Mines, played an essential role from the beginning. Other researchers involved in the study include Varda Lev-Ram of UCSD's department of pharmacology; Chris Schaffer of UCSD's department of physics; Roger Tsien of UCSD's departments of chemisty and pharmacology and the Howard Hughes Medical Institute; Qing Xiong of HHMI; and Agustin Ifarraguerri and Beverly Thompson of Science Applications International Corp.

The project was funded by the National Institute of Health's National Institute of Neurological Disorders and Stroke, the National Science Foundation and the David and Lucile Packard Foundation. The university has applied for a patent on the new technique.

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