The main improvements to immunofluorescence lie in the development of fluorophores and fluorescent microscopes. Fluorophores can be structurally modified to improve brightness and photostability, while preserving spectral properties and cell permeability.
Super-resolution fluorescence microscopy methods can produce images with a higher resolution than those microscopes imposed by the diffraction limit. This enables the determination of structural details within the cell. Super-resolution in fluorescence, more specifically, refers to the ability of a microscope to prevent the simultaneous fluorescence of adjacent spectrally identical fluorophores (spectral overlap). Some of the recently developed super-resolution fluorescent microscope methods include stimulated emission depletion (STED) microscopy, saturated structured-illumination microscopy (SSIM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM).