Breaking the Diffraction Barrier
Scientists used to view the diffraction barrier as a hefty obstacle to seeing further inside cells. However, in the past few years, a flurry of technical advancements have improved the resolving power of fluorescence microscopy by quantum leaps. Collectively known as “super-resolution” imaging, these methods are poised to provide biologists with unprecedented images of fine cellular structures and their dynamics inside the cell.
Two general strategies for breaking the diffraction barrier have developed independently. The first, called STED microscopy, works by the modulation of fluorophores - chemicals that can re-emit light upon excitation - by patterns of light inside a diffraction-limited region. In the second technique, applied in both PALM (short for Photo-Activated Localisation Microscopy) and STORM (3D Stochastic Optical Reconstruction Microscopy) microscopy, single-molecule imaging techniques are used to measure the position of individual molecules within a diffraction-limited region. Both methods take advantage of the ability to switch fluorophores between “on” states (in which light is emitted) and a “off” states (or dark states) by illuminating them with light at particular wavelengths.
In the image above, rat primary hippocampal neurons are visualised using STORM microscopy. The neurons are stained in two distinct colors - green-yellow and purple-pink. The shade of colour encodes the position on the z-axis, or height of the molecule - effectively allowing us to visualise neurons in three-dimensional space!