SUPER-RESOLUTION MICROSCOPY: BREAKING THE DIFFRACTION BARRIER
An exciting advance in recent years has been the development of super-resolution microscopy techniques that break the diffraction barrier and increase the resolution of fluorescence microscopy to the range of 10-100 nm, about tenfold less than the theoretical limit of resolution of the light microscope (see Figure 1.25). Several methods of super-resolution microscopy use fluorescent probes that shift the limit of resolution from the wavelength of visible light to the molecular level.
Figure 1.36 Super-resolution micro scopy (A) In conventional fluorescence microscopy, all of the fluorescent molecules in a sample fluoresce at the same time, yielding a blurred image. In STORM, only a small random fraction of the probes are fluorescent at any one time. Multiple images are obtained over time such that the individual fluorescent molecules can be resolved from one another and a super-resolution composite image is constructed. (B) Comparison of conventional and STORM microscopy of microtubules.
A good example is provided by the method known as STORM (stochastic optical reconstruction microscopy), which was developed in 2006 and has a resolution of approximately 20 nm. The principle underlying STORM and related methods is to produce high resolution by compiling individual images from thousands to millions of individual fluorescent molecules (Figure 1.36). In standard fluorescence microscopy, all of the fluorescent probes in a sample fluoresce at the same time. The fluorescent images of the individual molecules overlap to yield a blurred image with resolution limited by the diffraction of light. In contrast, STORM utilizes fluorescent probes that can be switched between the dark and fluorescent states. At any one time, only a small random fraction of the probes are fluorescent, such that the individual fluorescent molecules can be resolved from one another. Capturing many such images over time yields a series of snapshots, with different individual molecules emitting light in each picture. These multiple images can then be used to construct a composite image whose resolution is limited by the precision with which each fluorescent molecule is located, rather than by the diffraction of light.
The methods of super-resolution microscopy have been expanded to include three-dimensional imaging, simultaneous imaging of multiple different molecules using distinctly colored fluorescent probes, and super-resolution imaging of live cells. In addition to cytoskeletal structures, super-resolution microscopy has revealed fine-detailed structures of the nucleus, chromatin, sites of cell attachment, and the plasma membrane. It is likely that future research will result in the development of methods with even higher resolution than those available at present, allowing visualization of molecular events within living cells.