The detail that can be seen by optical microscopy is limited to ~0.25 microns (about 0.25 percent the diameter of a human hair). Although this level of detail can resolve some of the organelles of a cell, it falls short of revealing all the details of an organelle and is even less able to show any nanometer-sized structure that is responsible for so much of molecular biology. Combining the three insights has led us to a new kind of optical microscopy that can peer into this regime:
Single fluorescent molecules (which might label a protein of interest) can be imaged and their centers can be localized to a fraction of the size of the fuzzy spot that corresponds to their image.
Closely spaced, optically overlapping fluorescent molecules can be separated, and each can be localized if there is a distinguishing characteristic. For example, if two molecules light up separately in different image frames, the center of each can be inferred to a fraction of the spot sizes.
A new class of activatable fluorescent proteins has been developed in the past several years, and a distinguishing subset of these proteins can be turned into a fluorescing state while the remainder remains dark.
If we draw only the centers (and not the whole fuzzy ball) of all these fluorescent molecules that have each been imaged individually, we can generate a high-resolution image. The images in Figure 1 show a fluorescent protein–labeled Golgi apparatus in a cell seen by regular microscopy and by PALM.