A new microscopy technique has opened a window to the fast-moving molecular activity in living cells in real time – right down to the millisecond. Using a laser light sheet, researchers at the European Molecular Biology Laboratory in Heidelberg, Germany, scanned samples layer by layer, then reconstructed them in 3-D, providing insights into processes that previously were invisible. The new microscope’s illumination lens is shown from the side of the detection lens. The combination of light-sheet microscopy and single-molecule spectroscopy allows scientists to record the fluorescence of every pixel within view, capturing images at intervals of less than 1 ms. The setup includes an illumination unit that generates a laterally confined and thin diffraction-limited light sheet, a detection unit that contains a camera with single-molecule sensitivity to observe the focal region of the light sheet – referred to as the light pad – and an inverted microscope that allows for easy positioning of specific areas of the specimen in the light pad. The researchers used the microscope for fluorescence correlation spectroscopy to visualize the diffusion and interactions of proteins in mammal cells and in isolated fly tissue. The instrument enables observation of the way molecules diffuse across a whole sample, even if it contains several cells. Other techniques, based on confocal microscopy, allow visualization of only a few isolated spots in a sample at a time. “We can follow fluorescently tagged molecules in whole live cells, in 3-D, and see how their biochemical properties, like interaction rates and binding affinities, vary throughout the cell,” said Malte Wachsmuth, who developed the microscope. A new microscope setup follows the actions of single molecules in living cells in 3-D right down to the millisecond. Images courtesy of European Molecular Biology Laboratory/H. Neves. The tool holds promise for investigating a variety of processes, including the role of growth hormones in cancer, the regulation of cell division and signaling, and the patterning of tissue development in the embryo. Until now, chromatin – the combination of DNA, RNA and proteins that form chromosomes – was observed in either of two forms: the heterochromatin state, where it is wound together tightly, with most of the DNA inaccessible to the cell’s gene-reading mechanism, or the euchromatin state, where it is loosely packed and easily readable. Microscope components combine to bring molecular processes into focus. Using the microscope to measure the interaction between chromatin and a protein called HP1-α provides the team with a broader perspective. “In some areas that look like euchromatin, HP1-α behaves as it would in the presence of heterochromatin,” said Michael Knop, a researcher on the team. “This suggests that chromatin may also exit in an intermediate state between hetero- and euchromatin, which was not observable before in living cells.” The research was published online Aug. 7 in Nature Technology.