page_title: Super-resolution microscopy
section_title: Spatially modulated illumination (SMI)
hierarchical_section_title: Super-resolution microscopy / Super-resolution techniques / Spatially modulated illumination (SMI)
caption_attribution_description: English: Investigation of AMD,macular degeneration a medical condition which usually affects older adults and results in a loss of vision in the center of the visual field (the macula) because of damage to the retina macular degeneration. Sections from human eye tissue were analyzed with Structured Illumination Microscopy (SMI)/ Optical nanoscopy using a specially designed Super Resolution Microscope setup: LSI-TIRF as a total internal reflection interferometer with laterally structured illumination. This SMI technique allowed to acquire light-optical images of autofluorophore distributions in the tissue with previously unmatched optical resolution. Use of three different excitation wavelengths (488, 568 and 647 nm), enables to gather spectral information about the autofluorescence signal. Detailed Image description: Overview image. The SMI image (right) shows more details and less out of focus light than the widefield image (left). The Bruch membrane (B) is located between the choroid (C) and the retinal pigment epithelium (RPE). On the top of the image endings of retinal rod cells (R) can be seen. The scale bar is 2�m. Reference: Best G, Amberger R, Baddeley D, Ach T, Dithmar S, Heintzmann R and Cremer C (2011). Structured illumination microscopy of autofluorescent aggregations in human tissue. Micron, 42, 330-335.
context_page_description: Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.
  There are two major groups of methods for super-resolution microscopy in the far-field that can improve the resolution by a much larger factor:
  Deterministic super-resolution: the most commonly used emitters in biological microscopy, fluorophores, show a nonlinear response to excitation, which can be exploited to enhance resolution. Such methods include STED, GSD, RESOLFT and SSIM.
context_section_description: One implementation of structured illumination is known as spatially modulated illumination (SMI). Like standard structured illumination, the SMI technique modifies the point spread function (PSF) of a microscope in a suitable manner. In this case however, "the optical resolution itself is not enhanced"; instead structured illumination is used to maximize the precision of distance measurements of fluorescent objects, to "enable size measurements at molecular dimensions of a few tens of nanometers".
  The Vertico SMI microscope achieves structured illumination by using one or two opposing interfering laser beams along the axis. The object being imaged is then moved in high-precision steps through the wave field, or the wave field itself is moved relative to the object by phase shifts. This results in an improved axial size and distance resolution.
  SMI can be combined with other super resolution technologies, for instance with 3D LIMON or LSI-TIRF as a total internal reflection interferometer with laterally structured illumination (this last instrument and technique is essentially a phase-shifted photon tunneling microscope, which employs a total internal reflection light microscope with phase-shifted evanescent field (Guerra, 1996)). This SMI technique allows one to acquire light-optical images of autofluorophore distributions in sections from human eye tissue with previously unmatched optical resolution. Use of three different excitation wavelengths (488, 568, and 647 nm), enables one to gather spectral information about the autofluorescence signal. This has been used to examine human eye tissue affected by macular degeneration.
caption: SMI + TIRF of human eye tissue affected by macular degeneration
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