3D Nanoscopy: Bringing Biological Nanostructures into Sharp Focus

Abstract

Understanding the nanometer-scale organization of living cells, tissues, and materials is an essential goal of present and future research efforts, and yet zooming into the small compartments of life remains one of the great challenges of science. Even though microscopy techniques such as electron microscopy can help us, these approaches are usually limited by the elaborate and destructive sample preparation and measurement, or by the inability to study details beyond the surface of the sample. Fluorescence microscopy, on the other hand, has proven to be one of the most convenient and widespread tools to study key issues in the life sciences, though the problems that it can address are still limited. As with any microscopy technique, there is a lower limit to the sizes of the details that can be discerned. This finite resolution is a direct consequence of the wavelike character of light, known as the diffraction limit, which is about 250 nm in optical microscopy. This means that it is not possible to discriminate between two objects that are separated by less than that distance, and thus it seems that the nanostructuring of cells and materials is not accessible by direct optical means. More specifically, (part of) the reason that the resolution is limited is because a fluorescent “point object” of negligible size (such as a single molecule) does not appear as a point after imaging through the microscope but rather appears as a three-dimensional shape of much larger dimensions (Figure 1). Because the distributions from adjacent molecules in close proximity overlap we lose the ability to distinguish between molecules that are close together, and hence to visualize very small details of the sample. Several schemes recently developed to address these limitations have succeeded in providing images with unprecedented detail. In particular, several papers appeared in the last half of 2006 that converged into a similar approach, which has become known as photoactivation–localization microscopy (PALM). PALM relies on the insight that individual, well-isolated emitters can be localized with nanometer precision simply by determining the centroid of the emission distribution (Figure 1). If we could determine the position of all the emitters in this way then we could construct an image of the sample with nanometer resolution by carefully processing the fluorescence image. Unfortunately, the obtained data is meaningful only if we are indeed looking at the emission of a single isolated emitter (Figure 1), yet if we are interested in imaging beyond the diffraction limit then our sample likely contains many fluorophores within nanometers of each other. As a result, the only way to apply this localization procedure to practical samples is to separate the emission of the fluorophores in time, so that only a single fluorophore emits within a diffraction-limited region at any instant. In PALM this is achieved by making use of photoactivation: the stochastic “on” switching of the fluorescence emission at the single-molecule level followed by subsequent photobleaching or other deactivation. Hence PALM measurements are based on repeating cycles of on/off switching events, where fluorescence can be registered only for those molecules that are in the “on” state during a particular cycle (Figure 2). Because the activation of the fluorescence is a stochastic process, different molecules will be activated in each cycle, and if these molecules are spaced sufficiently apart then we can reconstruct the high-resolution picture. The need to control the fluorescence emission of the sample, down to the single-molecule level, through photoirradiation has recently inspired many studies and developments of photoswitchable fluorescent proteins and other compounds, though the method was applied mainly to fluorescent proteins Figure 1. The effect of diffraction: the original pointlike fluorescence emitters appear as a blurred distribution after imaging through the microscope. The positions of the isolated emitters can be recovered by determining the center-of-brightness of the resulting spots (indicated by the markers), although molecules that are spaced closer together than the diffraction limit cannot be separated (the blue marker and blue circle).