Super-resolution Fluorescence Microscopy

Conventional fluorescence microscopy can only resolve structures farther apart than 200 nm. This is because under a microscope, the diffraction of light makes the fluorescent molecules appear as blurry spots instead of sharp points. As a result, when objects are very close to each other, their images overlap.

Therefore, scientists have developed super-resolution techniques that provide better resolution to image even small subcellular structures. These techniques overcome the diffraction limits by using specific patterns of light to excite the fluorophores in the sample.

For instance, structured illumination microscopy uses a striped pattern which is then rotated to capture multiple images. The images are combined, creating an interference pattern that is computationally processed to make the final image.

Stimulated emission depletion microscopy creates a pattern using two lasers. A primary laser excites the sample while a donut-shaped beam suppresses the fluorescence around it, making the fluorescent point appear smaller.

Other techniques avoid overlapping signals by using fluorescent probes that can be switched on and off. Only some probes are activated for each image, minimizing signal overlap and allowing for the localization of a single fluorescent molecule. 

Many of these images can be combined into a single high-resolution composite image.