Super-resolution microscopy

Super-resolution microscopy, in light microscopy, is a term that gathers several techniques, which allow images to be taken with a higher resolution than the one imposed by the diffraction limit. Due to the diffraction of light, the resolution in conventional light microscopy is limited, as stated (for the special case of widefield illumination) by Ernst Abbe in 1873. In this context, a diffraction-limited microscope with numerical aperture N.A. and light with wavelength λ reaches a lateral resolution of d = λ/(2 N.A.) - a similar formalism can be followed for the axial resolution (along the optical axis, z-resolution, depth resolution). The resolution for a standard optical microscope in the visible light spectrum is about 200 nm laterally and 600 nm axially. Experimentally, the attained resolution can be measured from the full width at half maximum (FWHM) of the point spread function (PSF) using images of point-like objects. Although the resolving power of a microscope is not well defined, it is generally considered that a super-resolution microscopy technique offers a resolution better than the one stipulated by Abbe.Comparison confocal microscopy – 3D-SIMCell nucleus in prophase from various anglesTwo mouse cell nuclei in prophase.mouse cell in telophase Super-resolution microscopy, in light microscopy, is a term that gathers several techniques, which allow images to be taken with a higher resolution than the one imposed by the diffraction limit. Due to the diffraction of light, the resolution in conventional light microscopy is limited, as stated (for the special case of widefield illumination) by Ernst Abbe in 1873. In this context, a diffraction-limited microscope with numerical aperture N.A. and light with wavelength λ reaches a lateral resolution of d = λ/(2 N.A.) - a similar formalism can be followed for the axial resolution (along the optical axis, z-resolution, depth resolution). The resolution for a standard optical microscope in the visible light spectrum is about 200 nm laterally and 600 nm axially. Experimentally, the attained resolution can be measured from the full width at half maximum (FWHM) of the point spread function (PSF) using images of point-like objects. Although the resolving power of a microscope is not well defined, it is generally considered that a super-resolution microscopy technique offers a resolution better than the one stipulated by Abbe. Super-resolution imaging techniques rely on the near-field (photon tunneling microscopy as well as those that utilize the Pendry Superlens and near field scanning optical microscopy) or on the far-field. Among the latter are techniques that improve the resolution only modestly (up to about a factor of two) beyond the diffraction-limit like the confocal microscope (with closed pinhole), or confocal microscopy aided with computational methods such as deconvolution or detector-based pixel reassignment (e.g. re-scan microscopy, pixel reassignment ), the 4Pi microscope, and also structured illumination microscopy technologies like SIM and SMI. There are two major groups of methods for super-resolution microscopy in the far-field that can improve the resolution with a much larger factor: On October 8, 2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, W.E. Moerner and Stefan Hell for 'the development of super-resolved fluorescence microscopy,' which brings 'optical microscopy into the nanodimension'. In 1978, the first theoretical ideas had been developed to break the Abbe limit using a 4Pi microscope as a confocal laser scanning fluorescence microscope where the light is focused ideally from all sides to a common focus that is used to scan the object by 'point-by-point' excitation combined with 'point-by-point' detection. Some of the following information was gathered (with permission) from a chemistry blog's review of sub-diffraction microscopy techniques Part I and Part II. For a review, see also reference. In 1986, the super-resolution optical microscope based on stimulated emission was patented by Okhonin.