Super-Resolution Microscopy

Single-Molecule Localization Microscopy

In fluorescence microscopy, the diffraction of light causes individual fluorescence molecules to appear as fuzzy spots. If molecules are close together, these spots overlap, making them impossible to distinguish. This limits the resolution of an optical microscope to around half the wavelength of the fluorescence emission. To overcome this limit, we can use blinking fluorescence molecules. They flicker on and off at different times, allowing a computer program to find their center of gravity. The center of each fluorescence molecule can then be used to build a new image with greatly increased resolution.

Achieving this requires strong excitation light and proper chemical treatment of the sample. The tight light confinement and high-power laser unit make ACP-ZERO a highly effective imaging platform for these methods.

The figure above demonstrates how it is possible to obtain structural information based on stochastic photoswitching of fluorophores, essentially separating the fluorescent signal in time and space. This also means an optimized labeling density will be a critical consideration for the sample preparation.

a) Four fluorophores are depicted as white dots, each surrounded by a red glow. The four fluorophores are “on” and emitting, resulting in a blurry point. b) Blinking occurs when the fluorophore is cycled between an “on” state and an “off” state. This process is stochastic. The “on” state is indicated by a white-filled circle, while the “off” state is a dotted white circle. c) The center of gravity is then fitted computationally, resulting in a list of locations. d) The locations are then summed up, and the final image is reconstructed, with super-resolution.