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RESEARCH PROFILES Analyzing lone lipoproteins gree of saturation of the triglyceride-rich The original research goals of the Lipoproteins have always had to be examgroup were to detect individual proteins lipoprotein particles in blood samples. ined in bulk. They are the particles reOne difficulty with the method is on the surface of very low density liposponsible for carrying triglycerides and that the Raman signal from a single parprotein particles and to monitor how fatty acids around the body, and they ticle is very weak, so a long exposure these proteins changed in the blood become in multiple types, some of which fore and after a human subject ate a con- time of several minutes is needed. Chan play an important role in forming the and colleagues had wanted to monitor trolled-composition meal. However, the plaques that characterize heart disease. A the interaction of the lipoproteins with Raman spectrum was filled with noise— technique to analyze them individually in the enzymes in the endothelial cell their native environment could illu(b) walls. These enzymes facilitate the minate just what makes one lipopro30 -µL drop of TGRL Single, trapped breakdown of saturated fats. “If you tein plaque up and another harmless. plasma solution TGRL particle wanted to take one of these partiResearchers at Lawrence LiverGlass coverslip cles, bring it towards the endothelial more National Laboratory (LLNL); Translation stage layer, and monitor the biological the University of California, Davis; process in real time, you couldn’t,” and the U.S. Department of Agricul100 microscope with current techniques, says Chan. ture in Davis have adapted a hybrid objective His group is currently investigating technique to examine lipoproteins 633 -nm nonlinear methods of obtaining individually in solution. Described laser beam Raman spectra on time scales of in the September 15 issue of Anamilliseconds to seconds. lytical Chemistry (pp 5870–5786), The researchers have an ongoing the technique uses laser tweezing (a) CCD camera collaboration with oncologists at the to manipulate individual lipoproMicroscope university’s medical center who are tein particles and Raman spectrosobjective adapting the technique to flow cycopy to analyze the specific moletometry, to examine and sort cells cules that constitute the lipoprotein. The researchers initially develSpectrometer without damaging them. “If you can just look at the intrinsic [Raman] oped the method to examine triglycLaser signature, you could reuse the normal eride-rich lipoproteins (TGRLs), cells,” Chan says. Most traditional but it can be used to isolate and cell-sorting techniques use dyes to disanalyze any translucent particle or Schematic of (a) the confocal microscopy system used cell. After the laser is focused, a in laser-tweezers Raman microspectroscopy and (b) the tinguish cells from one another. They give a clear look—but tend to damage drop of solution containing lipopro- optical trapping of a single TGRL. or kill the cells in the process. In comteins is placed on a glass slide. The parison, laser-tweezers Raman microspecRaman signals from the diverse proteins researchers use a regular camera to troscopy uses a laser as a microscopic and lipids on the surface and within the watch for scattering of the laser light, the tractor beam. Nothing touches the cell or particle—which made it impossible to sign that a lipoprotein is trapped in the analyze the particular proteins of interest. particle but a harmless beam of light. The beam. The 633-nm trapping laser does collaborators recently submitted a paper double duty—it also excites the molecules “But it’s sort of a bonus,” says Chan. that describes the use of the technique “It’s good because you can get a large of the lipoprotein particle to give off snapshot of the entire biochemical prop- to distinguish healthy cells from cancerRaman signals, which are recorded by ous cells in certain forms of leukemia. erties of all the organic molecules and a standard Raman spectrometer. The researchers are also continuing compare it with another particle. The Raman spectroscopy has previously more info you get, the more discriminat- their work with very low density lipobeen used to analyze biological particles, proteins. “Cardiovascular disease is a ing power you have.” Despite the noisibut only after they were extracted from huge problem, and finding a technique ness of the data, the researchers could the sample, purified, and dried. A single dried lipoprotein is too tiny to reliably lo- clearly see the breakdown of saturated fats that could shed light on the cardiovascular disease process is very exciting,” in the lipoproteins from triglycerides into cate on a slide. “The initial novelty was says Chan. “I’m very interested in this free fatty acids. In samples taken 3 h after using Raman to study single biological translational research between physics particles of interest. All the previous stud- a meal was consumed, the researchers correlated the ratio of polyunsaturated fat and biology.” a ies have done bulk analysis,” says James —Kim Krieger to saturated fat in the meal with the deW. Chan, a physicist at LLNL. 372 A
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