What are the factors to consider when choosing a super-resolution technique for my experiment?

There are multiple factors to consider when choosing a super-resolution technique. One factor is the choice of probes, which function to enhance the image quality. These probes must possess the ability to switch reversibly or irreversibly between a light and dark state or to switch between different wavelengths. An ideal probe should be very bright, and exhibit a high contrast ratio between the light and dark states. It is important to note that choosing fluorescent probes is only a factor for microscopes which require them. Structured illumination microscopy for example does not require specialized probes since it is the most similar to fluorescent microscopy. 

Another factor is resolution, which refers to the smallest distance between two distinguishable points. The resolution of super-resolution methods are highly variable. For example, pixel reassignment methods generate a 1.4X improvement in resolution down to 120 nm, while the MINFLUX method has a resolution down to 1 nm. Multicolor imaging is another factor to consider, as SIM, pixel-reassignment methods, fluctuation-based methods, and ExM are all compatible since they can use different fluorophores. Multicolor imaging can be challenging in single molecule localization such as PALM. It has specific requirements and is limited to two-color experiments. Sample thickness is another factor, as thicker samples may experience out-of-focus light. When imaging deepers within these samples, there is an increase in spherical aberration due to differences in refractive indices, which degrades the image quality. An additional factor is the labeling of antibodies. Sometimes, multiple antibodies can attach to a single protein of interest and create various detected locations for each labeled molecule of that protein.