Scientists have developed a light microscope so powerful that it allows users to see inside cells and discern the precise location of nearly each individual protein they are studying.
Conventional optical microscopes are inherently limited by the wavelength of light, meaning. Objects separated by less than 200 nanometres cannot be distinguished from one another. The new approach, called photoactivated localisation microscopy (PALM), can discriminate molecules that are only two to 25 nanometres apart.
The researchers employed spectroscopic near-field microscopy, where a spectrum of light is spread out across wavelengths. This was used alongside molecular biology techniques that allow scientists to attach a new type of fluorescent label to each copy of a protein they wish to study.
The researchers label the molecules they want to study with a photoactivatable probe, and then expose those molecules to a small amount of violet light. The light activates fluorescence in a small percentage of molecules, and the microscope captures an image of those that are turned on until they bleach. The process is repeated approximately 10,000 times, with each repetition capturing the position of a different subset of molecules.
Because the number of molecules captured in each image is small, they are far enough apart to see each molecule individually and thereby localise its centre. When a final image is created that includes the centre of each individual molecule, it has a resolution previously only achievable with an electron microscope. Unlike electron microscopy, however, the new technique allows for more flexibility in labelling molecules of interest.