When the object you are imaging requires to go under the diffraction limit of 200 nm, this cannot be achieve using conventional microscope techniques. Then you need to look at the five popular super-resolution techniques: STED, STORM, SIM, PALM and 4Pi. All of them requires complex samples preparation, expansive microscopes and expert software-assisted analyses.
One the other way, it is now possible to achieve very good resolution using TIRF (Total Internal Reflection Fluorescence) microscopy coupled with SRRF (Super-resolution radial fluctuations) processing.
SRRF processing on microtubules
SRRF is an algorithm based on the principle that
noise in microscopy images fluctuates in time whereas fluorophores signal is stable.
SRRF algorithm uses image sequences acquired at high speed and combines spatial and temporal information to generate super-resolved images with a resolution close to 60nm (Culley et al,, 2018).
Traits computed with SRRF
All traits that an be extracted from images sequences acquired at high-speed with commercial confocal and widefield microscopes.