In 1873 Ernst Abbe defined the so-called "diffraction barrier": the spatial resolution limit attainable with conventional light microscopy as follows. Resolution is the light wavelength divided by twice the numerical aperture of the lens. A reasonable approximation to that is the full width half maximum (FWHM) of the point spread function of an optical system; the image of a point source of light. For example, a widefield microscope with a high numerical aperture (NA) objective (eg. 1.4), using a wavelength within the visible spectrum (eg. 550 nm), would give a resolution of about 200 nm.
However, in recent years different light microscopy techniques have been developed to work around this limit and to image and resolve features smaller than the diffraction barrier.
Here you can find more about the history and technical concepts of superresolution.
In this lecture, Prof. Xiaowei Zhuang surveys a variety of recent methods that achieve higher resolution than is possible with conventional microscopy with diffraction-limited optics. These include different types of patterned illumination (e.g. STED and SIM microscopy) or techniques that build up an image by stochastically switching on single fluorescent molecules and localizing each molecule with high spatial precision (STORM, PALM, FPALM).
In the following, we aim to give you a brief overview of the superrresolution techniques accessible to you on the campus and lead you to further interesting readings about them.
One of the best known superresolution techniques is STimulated Emission Depletion (STED) microscopy. Besides the normal, exciting laser beam this techniques uses a second, doughnut shaped beam around the focus point with which to selectively deplete fluorphores. With this approach the actual excited area is minimized, thus increasing the achived resolution beyond the diffraction barrier.
As a result of the development of this technique, Stefan Hell was awarded the Nobel Prize in Chemistry in 2014.
Here, Stefan Hell, who invented many of the methods allowing a resolution beyond the diffraction barrier, gives an introduction to these super-resolution microscopy techniques, and a detailed discussion of two such techniques: STED (Stimulated Emission Depletion) and RESOLFT (REversible Saturable OpticaL Fluorescence Transitions).
Find here an introduction to the STED concept. Includes an interactive Java Tutoial.
This tutorial explains the principles of STED super-resolution microscopy - from the underlying photo physical processes to the integration into a confocal laser scanning microscope.
Picoquant explains STED and introduces their implementation of it.
Find out more about the fundamentals of STED.
Here you can find a very detailed explanation of the principles of PALM and its possible applications.
In this lecture Prof. Xiaowei Zhuang begins by explaining that the resolution of traditional light microscopy is about 200 nm due to the diffraction of light. This diffraction limit has long hampered the ability of scientists to visualize individual proteins and sub-cellular structures. The recent development of sub-diffraction limit, or super resolution, microscopy techniques, such as STORM, allows scientists to obtain beautiful images of individual labeled proteins in live cells. In Part 2 of her talk, Zhuang gives two examples of how her lab has used STORM; first to study the chromosome organization of E. coli and second, to determine the molecular architecture of a synapse:
Find here a basic description to the PALM concept. Includes an interactive Java Tutorial.
During their 20-years friendship, nobel prize winning Eric Betzig and Hess worked together and separately, in academia and industry, before eventually joining forces to develop the first super-high-resolution PALM microscope. They tell us the story of this journey and emphasize how their unusual and varied backgrounds provided the skills to complete the project: Betzig and Hess
Find here another basic description to the PALM concept. Includes an interactive Java Tutoial.
In confocal-like structured illumination systems such as Apotome, Optigrid, DSD, and indeed point scanning and spinning disk confocals, structured illumination is primarily used to increase image contrast only. In contrast to that, further benefits of even higher contrast and doubled resolution are possible using a 3D structured illumination pattern of light projected into the sample.
This causes Moiré patterns: Interference fringes between the structured light pattern and the sample fluorescence distribution. These diffraction limited patterns contain doubled resolution information by the mathematical trick of Heterodyning, a very common method in engineering and signal processing.
Multiple images are required such that the light covers all the sample over the image series.
Then, extracting the higher resolution information from the interference fringes, and applying a deconvolution with the system PSF results in a reconstructed image with very high contrast (particularly in the usually poor axial, z direction) and doubled resolution in all directions.
This provides nearly an order of magnitude more spatial information.
No special fluorescent dyes are and no special sample preperation is needed over and above that for any other high resolution method. 3D-SIM out-performs point scanning confocal in speed, sensitivity, contrast and resolution for samples where the 3D light pattern is not disturbed by the sample, such as bacteria, yeast, and mammalian cell monolayers and even thick sections when mounted in high refractive index media.
OMX stands for Optical Microscope eXperimental, and came out of the Sedat lab in California, with hardware and image reconstruction software licenses to Applied Precision for the DeltaVision range of microscopes, then bought by GE Healthcare.
The traditional microscope stand, built around a human's eyes and hands was discarded, and instead the microscope was redesigned around having multiple cameras and a fixed inverted objective lens and nano motion xyz sample stage. This was to optimise the OMX for modern high performance biological fluorescence microscopy: Simultaneous multicolour widefield imaging with deconvolution and specifically, very high performance 3D-SIM.
Ring TIRF is also available on the OMX as a module. OMX has a second generation patterned light engine called Blaze. This makes live cell imaging with 3D-SIM possible, compared to the first generation slow, unstable mechanical pattern generators.
Combining TIRF with 2D-SIM both increases contrast, and can double spatial resolution.