Over the course of the past several decades, a variety of imaging modalities in fluorescence microscopy have emerged as powerful tools for probing the spatial and temporal dimensions in fixed and living cells and tissues to uncover structural and dynamic information. Widefield, confocal, multiphoton, and related fluorescence techniques are able to retrieve data from a specimen at resolutions approaching the wavelength of light based on the fluctuating distribution of fluorescently labeled entities that are unevenly distributed throughout the lateral and axial planes. Limitations in optical resolution for the microscope were first discovered in the late 1800s by Ernst Abbe who, together with Lord Rayleigh, created the foundation for modern microscopy by linking the imaging process to diffraction theory. In essence, microscope resolution is restricted by the diffraction of light at the objective rear focal plane, which creates a diffraction barrier that dictates a maximum resolution of approximately 200 nanometers in the lateral (x,y) dimension and 500 nanometers in the axial (z) dimension, depending upon the objective numerical aperture and the average wavelength of illumination. The diffraction barrier stood as an obstacle to high resolution imaging for over 300 years until the first limited improvements in lateral resolution were achieved by laser scanning confocal microscopy in the 1980s followed by more significant increases in axial resolution using interference coupled with standing wave illumination methodology in the late 1990s.

Continued development of advanced imaging techniques during the last decade has yielded a number of exciting developments that are collectively termed superresolution microscopy and feature both lateral and axial resolution measured in the tens of nanometers or even less, far surpassing the diffraction barrier, to enable the observation of structural details with unprecedented accuracy. These techniques are based on either probing the specimen with spatially modulated illumination or involve switching fluorophores on and off sequentially in time so that the signals can be recorded consecutively. Thus, for an implementation referred to as structured illumination microscopy (SIM), increased resolution is obtained by discarding traditional Köhler illumination and replacing it with non-uniform excitation light patterns that feature sinusoidal intensity variations in one or more dimensions coupled with powerful image reconstruction techniques. The other superresolution methods rely on spatially or temporally modulating the transition between two molecular states of a fluorophore (such as switching between a bright and dark state) or by optically reducing the size of the point-spread function used in the excitation illumination. Among the techniques that improve resolution by PSF modification are stimulated emission depletion (STED) and ground state depletion (GSD) microscopy. Alternatively, superresolution can also be achieved by temporally modulated precise localization of single molecules using photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). A large number of variations and improvements on these techniques continue to be reported.
The optical configuration and point-spread function improvement for superresolution structured illumination microscopy (SR-SIM) are illustrated in Figure 1. The microscope configuration is presented in Figure 1(a) and consists of a laser source directed into the microscope optical train using a multimode fiber coupler. The laser light, which is scrambled before entering the microscope, is directed through a polarizer and a diffraction grating, and is then projected onto the specimen. Only the -1, 0, and +1 diffraction orders are focused onto the objective rear aperture, and these are collimated by the objective in the specimen focal plane where they interfere and generate a three-dimensional (lateral and axial) sinusoidal illumination pattern (Figure 1(b)). Fluorescence emission generated by illumination of the specimen is captured by a CCD camera system. The point-spread function for superresolution structured illumination is dramatically reduced (Figure 1(d)) compared to widefield fluorescence. As will be discussed below, the lateral resolution of SR-SIM approaches 100 nanometers or better, whereas the axial resolution is improved to approximately 300 nanometers.