Vertico spatially modulated illumination (Vertico-SMI) is the fastest light microscope for the 3D analysis of complete cells in the nanometer range. It is based on two technologies developed in 1996, SMI (spatially modulated illumination) and SPDM (spectral precision distance microscopy). The effective optical resolution of this optical nanoscope has reached the vicinity of 5 nm in 2D and 40 nm in 3D, greatly surpassing the λ/2 resolution limit (about 200 nm for blue light) applying to standard microscopy using transmission or reflection of natural light (as opposed to structured illumination or fluorescence) according to the Abbe resolution limit That limit (also known as the Rayleigh limit) had been determined by Ernst Abbe in 1873 and governs the achievable resolution limit of microscopes using conventional techniques.
The Vertico-SMI microscope was developed by a team led by Christoph Cremer, emeritus at Heidelberg University, and is based on the combination of light optical techniques of localization microscopy (SPDM, spectral precision distance microscopy) and structured illumination (SMI, spatially modulated illumination).
Since March 2008 many standard fluorescent dyes like GFP and Alexa fluorescent dyes can be used with this so-called SPDMphymod (physically modifiable fluorophores) localization microscopy, for which only one single laser wavelength of suitable intensity is sufficient for nanoimaging.
Configuration
SMI stands for a special type of laser optical illumination (spatially modulated illumination) and Vertico reflects the vertical arrangement of the microscope axis which renders possible the analysis of fixed cells but also of living cells with an optical resolution below 10 nanometers (1 nanometer = 1 nm = 1 × 10−9 m).
A particularity of this technology compared with focusing techniques such as 4Pi microscopy, is the wide field exposures which allow entire cells to be depicted at the nano scale. Such a 3D exposure of a whole cell with a typical object size of 20 µm × 20 µm require only 2 minutes. Wide field exposures signify that the entire object is illuminated and detected simultaneously.
Spatially modulated illumination
SMI microscopy is a light optical process of the so-called point spread function-engineering. These are processes which modify the point spread function (PSF) of a microscope in a suitable manner to either increase the optical resolution, to maximize the precision of distance measurements of fluorescent objects that are small relative to the wavelength of the illuminating light, or to extract other structural parameters in the nanometer range.
The SMI microscope being developed at the Kirchhoff Institute for Physics at Heidelberg University achieves this in the following manner: The illumination intensity within the object range is not uniform, unlike conventional wide field fluorescence microscopes, but is spatially modulated in a precise manner by the use of two opposing interfering laser beams along the axis. The principle of the spatially modulated wave field was developed in 1993 by Bailey et al. The SMI microscopy approach used in the Heidelberg application moves the object in high-precision steps through the wave field, or the wave field itself is moved relative to the object by phase shift. 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 SMI technique allowed to acquire light-optical images of autofluorophore distributions in the sections from human eye 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. This has been used for of human eye tissue affected by macular degeneration AMD.
SPDM: localization microscopy
A single, tiny source of light can be located much better than the resolution of a microscope: Although the light will produce a blurry spot, computer algorithms can be used to accurately calculate the center of the blurry spot, taking into account the point spread function of the microscope, the noise properties of the detector, and so on. However, this approach does not work when there are too many sources close to each other: The sources all blur together.