Superresolution imaging using single-molecule localization

Abstract

Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (approximately 10-20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-molecule-based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales.

Figure 1

The point spread function (PSF) and principle of single-molecule superresolution imaging. (a) Focused light through a lens always produces a blurred or diffracted spot that is commonly represented by the PSF (left panel) with dimensions of ∼200 nm laterally (x, y) and ∼500 nm axially (z) for green light imaged through a lens having a numerical aperture of 1.4. The lateral PSF of a fluorescent point emitter can be fitted to a two-dimensional Gaussian distribution (right panel) to obtain the centroid of the fluorescent probe with nanometric accuracy. This is a key principle of single-molecule superresolution: fitting with high precision the centroid of a diffracted, blurry spot from a fluorescent molecule. (b) Single-molecule superresolution in practice is achieved by illuminating a densely populated specimen with low-intensity activation light so that only a sparse pool of molecules is activated. The position of each molecule is localized by fitting the measured photon distribution with a two-dimensional Gaussian function. These molecules are then photobleached before additional cycles of activation and photobleaching of new molecules are performed. A composite superresolution image (see the panel marked “Summed”) that contains information about the localization of many single molecules can thereby be produced within a single diffraction-limited region. (c) PALM image of a thin section through a lysosome expressing CD63 tagged with the photoactivated fluorescent protein Kaede, revealing highly localized molecules on the membranes of this organelle. Figure adapted from Reference 10.

Figure 2

Localization precision, resolution, and molecular density in single-molecule localization techniques. This series of images of Bossy the cow demonstrates how the superresolution image is built by plotting localized molecules, depicted here as points. For the purposes of illustration, the point sizes in panels a–c are depicted much larger than would be necessary to achieve the final image shown in panel d. The image of Bossy in panel a shows many localized points, but the relatively low density does not allow recognition of the image. As more points are placed in the images in panels b and c, it becomes more recognizable, but distinguishing (resolving) the fine features associated with Bossy's image requires a higher density of points, as shown in panel d. Thus, an image can be produced that contains precisely localized points using single-molecule localization techniques, but the density of those points affects directly the capability to resolve the features of the image.

Figure 3

iPALM image showing the three-dimensional localization of αv-integrin tagged with td-EosFP in a U2OS cell. Single molecules are color-coded based on their z position (see the color scale in the panel a), with red molecules closest to the coverslip, followed by yellow, blue, and purple molecules at further distances. Displayed in both the top and side views, integrin molecules distribute at multiple cellular sites. In addition to their concentration within feet-like focal adhesions (FA) found near the coverslip (see the yellow molecules), they can be seen in the plasma membrane (PM, green molecules) and in the tubular network comprising the endoplasmic reticulum (ER, blue-purple molecules) positioned ∼100–200 nm up from the PM. Figure adapted from Reference 79.

Figure 4

Comparison of three-dimensional single-molecule localization techniques. The three-dimensional precision of iPALM and defocusing techniques is shown as a function of the number of photons emitted from gold beads. iPALM (filled circles and filled squares) has better localization precision per emitted photon than defocusing techniques (unfilled circles and squares) and displays better axial (filled red circles) than lateral (filled blue squares) resolution. Conversely, defocusing techniques have markedly less precision per emitted photon in the axial direction (unfilled red circles) compared with the lateral direction (unfilled blue squares). FWHM, full width at half-maximum. Figure redrawn from Reference 79.

Figure 5

Imaging dynamic, dense populations using single-particle-tracking PALM. The molecular motion of VSVG-tdEosFP was imaged in a COS 7 cell using 561-nm light while simultaneously photoactivating subsets of the tdEosFP molecules. (a) The molecules were localized in each frame, and their determined positions in consecutive frames were linked into tracks. These represent molecules that fluoresced for > 0.75 s and are plotted as different colors to distinguish individual tracks. (b) The diffusion coefficient for each track was determined and plotted as a filled circle at the start of the track. Each has been assigned a color based on its value of D (see color scale on right).