Unfortunately, light behaves badly in living cells — scattering frantically and going to the wrong pigments, where it becomes absorbed. This unruly behavior makes it hard to follow biochemicals in cells, especially in 3-D.Elaborate setups with several cameras, mirrors and beamsplitters have been used for high-resolution 3-D tracking, but now researchers at the University of Illinois at Urbana-Champaign have used a simpler setup to monitor motor proteins and melanin in 3-D in living cells. To do 3-D tracking, the researchers combined basic in-focus microscopy in 2-D with defocused imaging, which exploits the loss of focus that occurs in the third dimension. The loss of focus results in ring-shaped aberrations, the radii of which are directly related to the depth of the object of interest. The researchers’ experimental setup included an Olympus microscope and an Andor Technology CCD camera. A beamsplitter from an Optical Insights Dual-View imager sent the focused image to one half of the camera and the defocused image to the other half. The holder for the beamsplitter created distance between the camera and the microscope, putting the image out of focus. To focus part of the image, the researchers inserted a focusing lens into the holder. Because in vitro experiments have fewer confounding factors than cellular experiments, the researchers initially tracked chromophores outside of cells. They followed polystyrene beads moved in 3-D with a Mad City Labs piezoelectric stage, and they also imaged quantum dots. To account for the unique emission properties of quantum dots, they replaced the focusing lens with a cylindrical one. Next they monitored kinesin labeled with polystyrene beads as it walked on bundles of microtubules that were placed on molecular stairs created from an artificial polymer.In the first cellular experiment, the researchers followed fluorescent polystyrene beads with 50-ms temporal resolution as motor proteins moved the beads within cells, after the beads were phagocytosed. They said that this experiment demonstrated that their imaging method can elucidate motor protein regulation — an important facet of intracellular transport.Scientists tracked melanin-containing organelles in 3-D in melanocytes, achieving a time resolution of a couple milliseconds. The left and right images are defocused and focused, respectively. A melanin-containing organelle is circled and is shown in the inset.In the second cellular experiment, the researchers pursued organelles that contain melanin as they migrated within melanocytes, and they attained a 2.2-ms temporal resolution (see figure). The piezoelectric stage jostled the organelles while the cells were affixed to the stage.Thus, the researchers demonstrated that they could resolve objects as small as several nanometers with a temporal resolution of several milliseconds using only one camera, a microscope and a beamsplitter.Nano Letters, July 2007, pp. 2043-2045.