PHOTOACTIVATION LOCALIZATION MICROSCOPY
Photomicrography by Harald Hess, Janelia Farms Research Institute, HHMI
Images and text reprinted here in abbreviated form.
Three images illustrating the limitation of traditional light microscopy and the power of a super-resolution technique called Photoactivation Localization Microscopy.
When a microscope lens collects light from a molecule and reimages it onto a camera, the light forms a "blurry" spot that reflects the size of the light's wavelength instead of the molecule's size.
This is called "diffraction," and it restricts the resolution of light microscopy to ~1/2 the light's wavelength (~250 nm) or approximately the size of a small mitochondrion.
Thus, imaging with traditional light microscopy is like painting with a "blurry" brush. Individual molecules closer in space than ~250 nm will appear as one.
[1] Here a U2OS [human osteosarcoma] cell is labeled with fluorescent proteins and imaged with traditional microscopy using TIRF (total internal reflection) microscopy, with a resolution of ~250 nanometers.
[2] The same cell is now imaged with Photoactivation Localization Microscopy, shrinking the resolution from ~250 nm to 20 nm. Individual molecules of the membrane protein farnesyl labeled with a photoactivable fluorescent protein are distinguishable.
[3] The same cell is rendered using a high-speed version of the super-resolution technique. This allows real-time imaging in live cells. 3D images can be created by finding the axial position of each molecule with methods such as interferometry.
SOURCE: Zeiss Cell Picture Show