Super-resolution microscopy (SRM) describes a number of microscopy techniques that enable imaging of cellular structures at resolutions beyond the Abbe diffraction limit. This limit restricts the optical resolution in conventional light microscopy. Resolutions below the diffraction barrier reveal more details of cellular structures, which allows a better understanding of many biological processes.
In general, two main technical approaches are used in SRM:
- Deterministic super-resolution techniques like STED and GSD
- Stochastic super-resolution techniques like STORM, dSTORM, and PALM
Limitations of diffraction limited optical microscopy
The maximum resolution of cellular structures beyond the wavelength of illumination (visible light ranges from approximately 400 to 700 nm) in a modern optical microscope is roughly about 250 nm laterally and 600 nm axially. Fluorophores that are located within a few hundred nanometers within a cell can’t be distinguished and thus are detected as one blurred signal.
In 2014 the Nobel Prize in chemistry was awarded to Eric Betzig, Stefan Hell and William Moerner for the development of super-resolved fluorescence microscopy.PRODUCTS
Stochastic optical reconstruction microscopy (STORM)
STORM relies on the sequential activation and time-resolved localization of photo-switchable (or “blinking”) fluorophores to generate a high-resolution image. ChromoTek’s Nano-Boosters, Nano-Labels, and Nano-Secondaries, which consist of Nanobodies conjugated to fluorescent dyes, locate the photo-switchable fluorophore in proximity to the cellular target because of their small label to epitope displacement. ChromoTek offers STORM compatible nanoprobes, i.e. Nano-Booster, Nano-Label, and Nano-Secondaries®, which are conjugated to blinking fluorophores:
Alexa Fluor 488
Alexa Fluor 568
Alexa Fluor 647
Stimulated emission depletion (STED)
STED applies two laser pulses to localize fluorescence at each focal spot: The first laser pulse excites the fluorophore and the second depletes any fluorescent signals surrounding the excitation focal spot. The focal spot is raster-scanned across the sample to generate the high-resolution image.
ChromoTek offers STED compatible nanoprobes, i.e. Nano-Booster, Nano-Label and Nano-Secondaries®, which are conjugated to stable fluorophores:
Alexa Fluro 568
Abberior Star 635P