Super-resolution microscopy techniques are so-called because of their ability to resolve structures beyond the diffraction limit of light. Conventional light microscopy techniques are unable to bypass this limit, which prevents structures finer than roughly half the wavelength of the emission light (typically no less than ~200 nm) from being resolved. In contrast, super-resolution techniques have been shown to achieve single nanometer resolution.
Super-resolution imaging reveals more information about biological structures than conventional light microscopy by allowing smaller features to be visualized and in more detail. There are multiple types of super-resolution microscopy techniques, each with their own advantages and disadvantages.
Introduction to Super-Resolution Localization Microscopy
One family of super-resolution techniques are the super-resolution localization microscopy techniques, which comprise Photoactivated Localization Microscopy (PALM), Stochastic Optical Resolution Microscopy (STORM) and DNAbased Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT).
These techniques are able to temporally isolate single fluorophores from a group and take advantage of our ability to localize these fluorophores to sub-diffraction-limited spots. By individually localizing single fluorophores and reconstructing them into one image, a super resolved image can be genereated that effectively breaks the diffraction limit.
Introduction to SIM and iSIM
Structured Illumination Microscopy (SIM) and the high-speed alternative instant SIM (iSIM), can halve the resolution limit of conventional light microscopy.
However, unlike other super-resolution methods, iSIM can capture images at up to 100Hz, and with a greater degree of optical sectioning (rejection of out-of-focus light). This enables iSIM to generate 3D + time super-resolved images deep into biological structures, all while using conventional fluorescent dyes and molecules.
Introduction to Super-Resolution Spinning Disk Confocal
Spinning disk confocal microscopy is a versatile and widely used imaging technique in biology due to its ability to perform fast, 3D imaging of live cells.
Recently, techniques have been created that combine super-resolution imaging with the simplicity and optical sectioning capability of spinning disk confocal, resulting in a spinning disk system capable of a twofold resolution improvement over the diffraction limit.
Prime Scientific CMOS Camera Processing Tools for Super-Resolution Microscopy
The Prime Series of sCMOS cameras provide the highest levels of sensitivity which make them ideal for low-light imaging applications such as single molecule fluorescence and super-resolution microscopy.
The Prime cameras come equipped with in-line processing features which can be enabled to improve point localization performance for localization based super-resolution imaging. These processing features, PrimeEnhance™ and PrimeLocate™, can improve signal-to-noise ratios to increase localization accuracy as well as aiding with falsepositive localized events and data management.
Camera Comparison: Prime 95B sCMOS vs EMCCD
To support our claim that the Prime 95B is the best camera for super-resolution localization microscopy, we’ve collaborated with some of our customers to compare the localization accuracy of the Prime 95B to popular EMCCD cameras.
We also investigated the improvement made by PrimeEnhance™, the live denoising algorithm that accompanies the Prime 95B.
To perform these tests, DNA-origami nanostructures with a pre-defined pattern of fluorophores were provided by GATTAquant and used as precision standards.