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RESEARCH PROFILES Probing single molecules in living cells Anyone who has taken a biology course knows that cells are complicated creatures—filled with all kinds of molecules and structures, each with a name and function to memorize for the next exam. So some eyebrows might be raised by the report in the November 15 issue of Analytical Chemistry (pp 5606–5611) that Shuming Nie, Tyler Byassee, and Warren Chan at Indiana University have found and identified single molecules inside a living cell. Like the four-minute mile or the first spaceflight, this marks the breach of an important barrier. Before now, several research groups had detected individual macromolecules in solution or on the surface of a cell using fluorescence microscopes. The challenge when looking inside the cell is that the internal environment contains billions of fluorescent molecules as well as complex organelles and is known to produce intense background fluorescence. Thus, a major concern is that this intracellular background could overwhelm the relatively weak signals arising from single molecules. Further, molecules inside a living cell are either in constant motion or are attached to surfaces—perhaps even hidden. How can you chase down one of these molecules so you can identify it? Rather than pursue the molecule, the Indiana scientists wait for it to come by. They use a confocal fluorescence microscope to focus a laser beam on a selected spot inside the cell. The volume of the analytical spot is tiny—about 1 f L. Individual target molecules simply migrate through this volume. “The emissions from each passing molecule are recorded as bursts of photons,” Byassee explains. “The background fluorescence from inside the cell is noticeable, but is constant and much less intense than the bursts of photons resulting from a passing fluorescent probe molecule.” To en-
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those found in pure solvent. Single molecule data were obtained for three types of fluorescent molecules inside human HeLa cells. First, the iron transport protein, transferrin—labeled with tetramethylrhodamine, which is rapidly taken up by the cell by a process called receptor-mediated endocytoLaser beam sis—was detected inside living focused inside cell cells. Second, the cationic dye rhodamine 6G (R6G) entered cultured cells by a Objective (100X) potential-driven process, and single R6G molecules were observed as intense Objective (100X) photon bursts when they (a) (b) moved in and out of the focused laser beam. Third, the researchers found that certain synthetic oligonucleoObjective Objective tides (oligos), tagged with a (100X) (100X) fluorescent dye, were taken up Focus inside cytoplasm Focus inside nucleus by living cells via a passive pathway that does not involve endocytosis. “We envision that further developments will include real-time tracking (A) Laser beam focused inside a single living and two-color fluorescence measurecell by the confocal microscope. The beam can be focused (B) in the cytoplasm, or (C) in ment at the single molecule level,” says Nie. “Such capabilities will allow the the nucleus. direct observation of key intracellular events, such as the transport of gene scope is similar to that previously retherapy vectors, hybridization of antiported by William Lyon and Nie (Anal. sense oligos to mRNA, and ligand– receptor binding and internalization.” Chem. 1997, 69, 3400–3405). The James P. Smith and excitation source is a continuous-wave Vicki Hinson-Smith argon ion laser (514-nm emission), directed into the back port of an inverted microscope. The laser light reflects from a dichroic beam splitter and is focused onto the sample by a high numerical Raman studies bacteria aperture oil-immersion objective. Fluoone by one rescence photons are collected through the same objective and detected by an As the biotechnology industry grows, avalanche photodiode. bacteria are gaining importance as bioThe recorded peak heights are typicatalysts in various processes, including cally more than 3 times that of the the treatment of waste and the recovery background. Peak width is an indication of metals. One outcome of the increased of the diffusion rate of the fluorescing interest in such “bioprocesses” is the molecule passing through the focused need to characterize the metabolism and spot. Surprisingly, the diffusion rates production of bacterial cell populations inside the cell appear to be similar to to optimize their growth and performsure that the cells are still viable after the experiments, the researchers limit the laser exposures to ~12 s/run. The confocal fluorescence micro-
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