What is super resolution microscopy?
Super resolution microscopy (SRM) is an imaging technique that allows for the observation of biological structures at a higher spatial resolution than what is possible with traditional light microscopes. SRM can be used to obtain images with resolutions down to tens of nanometers, which is significantly smaller than the diffraction limit of light (~200 nm). This improved resolution is made possible by using special fluorescent probes that emit light at very specific wavelengths and by employing clever image processing algorithms.
While super resolution microscopy was first developed in the late 1990s, significant advances have been made in recent years that have greatly expanded its capabilities. For instance, newer SRM methods such as stimulated emission depletion (STED) and photoactivated localization microscopy (PALM) allow for even higher resolutions to be achieved. In addition, these newer methods are also capable of imaging larger areas and thicker specimens than what was previously possible. As a result, SRM has become an indispensable tool for biomedical researchers who wish to study the structure and function of cells and other small biological objects in unprecedented detail.
How does super resolution microscopy work?
The basic principle behind all SRM techniques is similar: they exploit the fact that when two or more point sources of light are brought closer together than the wavelength of the light they emit, their images will no longer appear as separate points but will instead start to overlap. By careful control of the position and intensity of these point sources, it is possible to create an image with sub-wavelength features. The most common way to generate such point sources is through the use of fluorescent dyes or proteins that can be selectively targeted to specific structures within a cell or tissue sample. Once these molecules are “tagged” with a fluorophore, they can be excited by laser light so that they emit photons at very specific wavelengths. When multiple fluorophores are present within close proximity to each other (< 200 nm), their emissions will begin to overlap and produce an interference pattern known as far-field fluorescence emission4 . This interference pattern can then be captured by an objective lens and converted into an image using specialized software algorithms5 .