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Normally when a fluorescent sample is illuminated using techniques like widefield or confocal microsopy, effectively all its fluorophore molecules will emit light simultaneously. For SMLM, a method is required to ensure only a few molecules emit in a single image, that those molecules do not emit in the next image and that a different set of molecules emits in subsequent images so that all the molecules can be localised with as little duplication as possible. The on/off switching of fluorophores between frames is often described as 'blinking', as seen in the GIF below. SMLM techniques mainly differ in how the blinking is achieved.

SMLM Techniques

Photo-activated

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Localization Microscopy (PALM) or Fluorescence Photoactivation Localization Microscopy (FPALM)

PALM (or FPALM) was the first SMLM technique to be described in papers by Betzig et al and Hess et al. The original implementation used a photoactivatable form of GFP (PA-GFP) which is normally in a nonfluorescent state, but which can emit green fluorescence after photoconversion with 405 nm light. Sparse subsets of PA-GFP fluorophores were coverted into their emissive state using 405 nm light. The molecules were imaged until they photobleached and then another subset was photoconverted. This process was then repeated for thousands of images. Photoactivatable forms of fluorescent proteins can be challenging to image because they only become fluorescent after conversion, which makes it difficult to set up experiments. Photoconvertible FPs can also be used. These emit light at different wavelengths depending on whether they have been activated. For example, mEos2 normally emits green light but emits red light after photoconversion. In the case of photoconvertible FPs the blinking is achieved by converting a subset of the fluorophores in each frame. It is also possible to use organic dyes rather than FPs. In this case the dyes would be 'caged' and therefore would  be in a non-emissive state that undergoes photoconversion.

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