ABSTRACT

The wavelength of light was long thought to impose a fundamental physical limit on the resolution that could be achieved with a light microscope. Whilst super-resolved single color images are useful for some applications, many biological questions rely on the knowledge of the relative positioning of multiple different proteins. Localization microscopy, as described so far, improves lateral resolution, but leaves the axial resolution essentially unchanged at approximately 600 nm. Sample preparation for photoactivated localization microscopy/stochastic optical reconstruction microscopy imaging shares many similarities to conventional fluorescence microscopy, with strategies such as immunofluorescence and transgenic expression being used. Background affects localization-based methods in two ways: it reduces both the localization accuracy and the detectability of events prior to localization. Refractive index mismatch and optical aberrations are generally going to negatively affect the accuracy of all 3D methods. Localization-based super-resolution microscopy is a surprisingly simple fluorescence microscopy technique which allows a dramatic improvement in resolution to be obtained with reasonably modest instrumental complexity.