bio sphere
A first CLASS microscope A new type of microscopy visualizes organelles without fluorescent labels.
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luorescent labeling of organelles is a popular method for studying changes in subcellular architecture. However, fluorescently labeled molecules can be toxic or affect cell function. With a new optical imaging technique called confocal light absorption and scattering spectroscopic (CLASS) microscopy, Lev T. Perelman and colleagues at Harvard University and Beth Israel Deaconess Medical Center in collaboration with scientists from Boston University and Bedford VA Medical Center can visualize organelles in living cells without fluorescent labels (Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17,255–17,260). CLASS microscopy combines confocal microscopy, which achieves highresolution optical slicing of cells, with light-scattering spectroscopy (LSS). LSS measures the spectra of light scattered by small particles. According to Perelman, the principle behind LSS is similar to how a rainbow forms. “In a rainbow, light from the sun is deflected and scattered by very small water droplets in the air,” he says. “A rainbow appears because your eyes see the light-scattering spectra of the water droplets. In LSS, we replace the sunlight with a lamp, the droplets with cell organelles, and your eye with a spectrometer.” Perelman and colleagues first collect light-scattering spectra from microme ter-sized areas within cells and then reconstruct the sizes of organelles in these regions by applying a version of the Mie theory, a mathematical model that relates the light-scattering spectrum of a particle to its size and refractive index. According to Daniel Palanker at Stanford University, this characteristic makes CLASS microscopy an appealing approach for imaging live cells, because it eliminates the need for exogenous staining. Instead, he says, “from spectroscopic analysis of the light scattered by living cells, the sizes and optical densities of otherwise invisible objects can be determined.” © 2008 American Chemical Societ y
Because confocal micros copy uses a pinhole to eliminate scattered light from all but a narrow focal plane, the combination of confocal microscopy with LSS enabled Perelman and colleagues to zero in on tiny regions within cells. “We collected the lightscattering spectra from a very small point inside the cell, and these spectra told us what 5 µm organelles were present in that spot,” Perelman says. “Then we moved the light source to Reconstructed CLASS microscopy images of three una different spot and collected treated human bronchial epithelial cells (top) and three those spectra, and so on, until similar cells treated with an agent that induces apoptoeventually we created a whole sis (bottom). (Adapted with permission. Copyright 2007 picture of the cell.” Unlike National Academy of Sciences, U.S.A.) standard confocal microscopy, LSS is not diffraction-limited and can
