Vertico-SMI

The Vertico SMI is a fluorescence microscope for three-dimensional image of cells in the nanometer range ( Super Resolution Microscopy ). In contrast to other approaches, the marking is done with normal fluorescent dyes such as GFP, Cy2 / 3, fluorescein, Alexa and Attofarbstoffen, based on the so-called blinking phenomenon. It is based on two microscope technologies, which were developed in 1996, the SPDM localization microscopy and structured illumination SMI. The effective optical resolution of this optical Nanoskopes reaches 5 nm and 40 nm in 2D in 3D and will be much better than the physical resolution limit of 200 nm, postulated by the law of Abbe 1873.

Configuration

" SMI " refers to a special type of optical laser illumination ( spatially modulated illumination, Spatially Structured lighting) and " Vertico " for the vertical arrangement of the microscope axis, which enables fixed cells, but also living cells with a three-dimensional effective optical resolution of 40 nanometers ( 1 nanometer = 1 nm = 1 × 10-9 m ) to analyze.

Basics

The microscope was developed by Christoph Cremer, Professor of Applied Optics and Information Processing at the University of Heidelberg / Institute of Molecular Biology. It is based on a combination of light optical techniques of localization microscopy ( SPDM, Spectral Precision Distance Microscopy) and structured illumination (SMI, Spatially Modulated Illumination). A special feature in contrast to focusing techniques such as 4Pi microscopy, the wide-field images, in which the entire sample is simultaneously illuminated and detected. This allows all the cells quickly take nanoscopically. For 3D images all such cells with typical frames of size 20 microns x 20 microns 2 minutes are needed.

The effective optical resolution of this optical nanoscope has reached a range of 5 nm in 2D and 40 nm in 3D, and therefore is substantially below the physical limit of 200 nm, which by the law of Abbe in 1873 as the physical limit, below which a light microscopic resolution is theoretically not possible, it has been postulated.

Spatially Modulated Illumination ( SMI)

Spatially Modulated Illumination ( SMI) is spatially structured illumination. The SMI microscopy is a light- optical method of the so-called point spread function engineering. Including methods are intended to modify the point spread function ( point spread function, PSF) of a microscope in a suitable manner, either to increase the optical resolution, the accuracy of distance measurements of dot -shaped, i.e., in comparison to the wavelength of the small to maximize fluorescent objects or to extract other structural parameters in the nanometer range.

When present at the Kirchhoff Institute for Physics of the University of Heidelberg have developed SMI microscope this is achieved in that the excitation intensity in the object space, in contrast to conventional widefield fluorescence microscopes is not homogeneous but spatially precise modulated by using two counter-rotating, interfering laser beams in the axial direction will. The principle of the spatially modulated wave field in 1993 by Bailey et al. developed. In the Heidelberg SMI microscopy approach, the object is moved in a highly precise increments by the wave field or it will be the wave field itself shifted ( phase). This results in an increase in the axial size and distance resolution.

SMI can with other super-resolution microscopy technologies, such as 3D LIMON or with LSI TIRF as total internal reflection fluorescence (TIRF ) interferometer with spatially structured illumination are combined. In combination with TIRF light optical images of autofluorescent ( self-luminous ) structures in the human ocular tissue can be recorded with unprecedented optical resolution using three different excitation wavelengths ( 488, 568 and 647 nm). The optical resolution is 100 nm in studies of macular degeneration of diseased human eye tissue.

Spectral Precision Distance Microscopy ( SPDM )

SPDM ( Spectral Precision Distance Microscopy ) is a light- optical method of fluorescence microscopy, with which to " optically isolated " particles (such as individual molecules) position, distance and angle measurements below the optical diffraction limit are widely available. " Optically isolated " means that at any given time only a single particle / molecule in a specified by the conventional optical resolution area (typically 200-250 nm in diameter ) is registered. This is possible if the particles located in such an area / molecules have different spectral labels (e.g., have different colors, or other usable variations in the light emission point ).

The possible structure with SPDM resolution can be specified by the smallest measurable distance between two in their spatial position specific " point-like " particles of different spectral marker ( " Topological resolution "). Simulations have shown that, under suitable assumptions on localization accuracy, particle density, etc., the " topological resolution " of a spatial frequency corresponds to that is a greatly increased optical resolution in the sense of the classical definition equivalent.

There is a localization microscopy, whereby an effective optical resolution is made possible, which is better by far than the conventional optical resolution ( 200-250 nm), given by the half width of the main maximum of the effective point spread function. By a suitable laser optical precision method positions and distances are considerably smaller the half- width of the point spread function between target objects with different spectral signatures as measured with nanometer precision. An important application is the genomics ( study of the functional organization of the genome ). Another major area of ​​application is the membrane structure research.

SPDMphymod " flashing colors " instead of photoswitchable molecules

Cremers Working Group in 2008 found that the super resolution microscopy can be realized under certain photophysical conditions for many " ordinary " dye molecules such as GFP or Alexa dyes. Characterized luminescent molecules can be used (but with a different spectral signature due to " blink" properties ) at the same spectral color. The combination of many thousands of individual shots of the same cell using laser optical precision measurements " localization images " obtained with significantly improved optical resolution.

This expands the applicability of the method on many areas SPDM biophysical, cell biological and medical research.

• Super Resolution Microscopy - Localization Microscopy

Two-color localization microscopy SPDMphymod / Super Resolution Microscopy with GFP and RFP fusion proteins

Marker / probe -free localization microscopy SPDM - previously unseen cell structures are detectable

LIMON: 3D Super Resolution Microscopy

LIMON (Light Microscopical nanosizing microscopy) was developed in 2001 at the University of Heidelberg and combines the two methods Localization Microscopy and Structured Illumination for 3D microscopy.

The procedure is as follows: first SMI shots are taken and then performed the SPDM process. The SMI process determines the center of the particles and their propagation in the direction of the microscopic axis. While the center of the particles / molecules can be determined with an accuracy of 1-2 nm, the spread in this point to an axial diameter of about 30-40 nm can be determined. Then the lateral position of the individual particles / molecules with SPDM with an accuracy of a few nanometers is determined. Currently SPDM reached 16 frames per second ( depending on the camera) with an effective resolution of 5 nm in 2D ( object level); about 2,000 such shots are combined with SMI data to achieve a three-dimensional image of the highest possible resolution (effective optical 3D resolution 40-50 nm).

The spatial arrangement of the two active genes in breast cancer and HER2/neu HER3 determined with an accuracy of about 25 nm, and their for carcinogenesis probably relevant clustering was analyzed at the single molecule level by this two-color Kolokalsiations 3D Super Resolution Microscopy.

Also Biomolecular machines, highly complex nanostructures that fulfill basic functions in the body cells, Resolution Microscopy LIMON can be studied studied in their biologically relevant composition with the 3D Super. Individual proteins or nucleic acids, which are hidden in the 3D molecular complex so-called biomolecular machines can be made visible without destroying the complex by variable mark.

Use of super resolution microscopy in the industry

In addition to the customary use of methods for super resolution microscopy in biomedical laboratories, these technologies can also make an important contribution in pharmaceutical research. Interactions of pharmaceutically active compounds with 3D molecular complexes so-called biomolecular machines (responsible for important processes in the body) can be tracked specifically in vivo and basic mechanistic questions are answered ingredients.

Of any practical use the super resolution microscopy can also be useful for quality management in the food and Agraindustrie. From this technology, a quality management would be developed, which allows a quick check of minutes in a series of pathogens such as the hospital bug MRSA, as well as the antibiotics detection example in poultry meat.