Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Superresolution Microscopy

Saturated Structured Illumination Microscopy

In the non-linear structured illumination techniques named saturated pattern excitation microscopy (SPEM) and saturated structured illumination microscopy (SSIM), the ground state (S0) is depleted by saturated excitation (to the S1 state) through an inverse RESOLFT scheme that enables the fluorescence generated by this transition to be recorded on an area-array detector. These techniques are performed using a grid-like array of line-shaped intensity maxima and minima rather than the typical doughnut-shaped phase modulation that is utilized by STED and GSD. The saturated excitation produces narrow line-shaped dark regions in the zero nodes that are surrounded by high levels of fluorescence signal to generate a "negative" imprint of the features being imaged. Thus, in SPEM and SSIM, it is the off state of a fluorescent probe (rather than the on state) that is confined by the phase modulation zero node. The grid lines are rotated several times to generate data for a single image, which is retrieved mathematically during post-acquisition processing.

In SSIM and SPEM, structured illumination is produced by two powerful interfering light beams from a single laser that form a standing wave grid pattern projected across the lateral dimensions of the specimen. Only the first diffraction orders (+1 and -1) are used to create the interference pattern and the zeroth order from the laser source is blocked with a beam stop. Most of the exposed fluorescent probes within the specimen saturate due to exposure to the intense illumination, leaving only narrow dark line-shaped volumes (zero nodes) having a period of approximately 200 nanometers at the edges of the interference pattern. As the excitation energy is increased, the walls surrounding the nodes become much larger, thus reducing the size of the point-spread function. The methodology is implemented on widefield, non-scanning microscopes by phase-shifting the pattern to render otherwise unresolvable superresolution information visible in the form of harmonic frequencies and fundamental moiré fringes. Due to the fact that resolution is only improved in the direction perpendicular to the line-shaped zero nodes, the pattern must be shifted in several directions to cover a large number of angles in the focal plane.

Non-linear structured illumination techniques are similar to STED microscopy in that the time to conduct measurements is theoretically independent of labeling density, and photostability of the fluorescent probes dictates performance. The mechanism by which resolution is increased in SSIM and SPEM is described in terms of Fourier components and spatial frequencies. However, similar to the other point-spread function engineering techniques of STED and GSD described above, non-linear structured illumination microscopy takes advantage of steady-state saturation where high resolution information is obtained only after the system has reached fluorophore-saturating conditions. Additionally, spatial resolution scales with the level of saturation to generate images having a lateral resolution in the vicinity of 50 nanometers under optimal conditions of signal-to-noise and low photobleaching. Because of the time necessary to rotate the projected grid pattern, the specimen must be motionless for relatively long periods of time, but the fluorophores suitable for SSIM and SPEM do not require specialized photoswitching properties.

Contributing Authors

Stephen P. Price and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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