Figure 1. Super-resolution imaging... | Rockefeller University Press

Figure 1.

$Figure 1. Super-resolution imaging principles. (A) In SIM the sample plane is excited by a nonuniform wide-field illumination. Laser light passes through an optical grating, which generates a stripe-shaped sinusoidal interference pattern. This combines with the sample information originating from structures below the diffraction limit to generate moiré fringes. The image detected by the CCD camera thus contains high spatial frequency sample information shifted to a lower spatial frequency band that is transmitted through the objective. A mathematical reconstruction allows, from a series of 15 raw images per slice, to reconstruct a high-resolution image with doubled resolution in xy compared with wide-field resolution. In 3D-SIM additional doubling in the axial resolution is achieved by accounting for an additional modulation introduced along the axial direction. (B) In STED microscopy the focal plane is scanned with two overlapping laser beams, typically being pulsed with a mutual time delay. While the first laser excites the fluorophores, the second longer wavelength laser drives the fluorophores back to the ground state by the process of stimulated emission. A phase plate in the light path of the depletion laser generates a donut-shaped energy distribution, leaving only a small volume from which light can be emitted that is then being detected. Thus, the PSF is shaped to a volume smaller than the diffraction limit. (C) Single molecule localization microscopy assures that only a relatively low number of fluorophores are in the emitting (active) state. This is achieved either by photoactivation, photoswitching, triplet state shelving, or blinking. These molecules are detected on the CCD camera as diffraction-limited spots, whose lateral position is determined with very high accuracy by a fit. Single molecule positions from several thousand raw images, each with a different subset of emitters, are then used to generate a density map featuring several hundred thousand single molecule positions within the plane of focus.$

Super-resolution imaging principles. (A) In SIM the sample plane is excited by a nonuniform wide-field illumination. Laser light passes through an optical grating, which generates a stripe-shaped sinusoidal interference pattern. This combines with the sample information originating from structures below the diffraction limit to generate moiré fringes. The image detected by the CCD camera thus contains high spatial frequency sample information shifted to a lower spatial frequency band that is transmitted through the objective. A mathematical reconstruction allows, from a series of 15 raw images per slice, to reconstruct a high-resolution image with doubled resolution in xy compared with wide-field resolution. In 3D-SIM additional doubling in the axial resolution is achieved by accounting for an additional modulation introduced along the axial direction. (B) In STED microscopy the focal plane is scanned with two overlapping laser beams, typically being pulsed with a mutual time delay. While the first laser excites the fluorophores, the second longer wavelength laser drives the fluorophores back to the ground state by the process of stimulated emission. A phase plate in the light path of the depletion laser generates a donut-shaped energy distribution, leaving only a small volume from which light can be emitted that is then being detected. Thus, the PSF is shaped to a volume smaller than the diffraction limit. (C) Single molecule localization microscopy assures that only a relatively low number of fluorophores are in the emitting (active) state. This is achieved either by photoactivation, photoswitching, triplet state shelving, or blinking. These molecules are detected on the CCD camera as diffraction-limited spots, whose lateral position is determined with very high accuracy by a fit. Single molecule positions from several thousand raw images, each with a different subset of emitters, are then used to generate a density map featuring several hundred thousand single molecule positions within the plane of focus.