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SMI = Spatially Modulated Illumination

General:

SMI microscope. The SMI microscopy is a new light-optical procedure using so-called POINT Spread Function engineering methods. Its procedures are those which modify the point image function (POINT Spread Function, PSF) of a microscope in an appropriate way, in order to either increase the optical resolution, to maximize the precision of distance measurements at punctiform objects (i.e. fluorescent objects which are small in comparison to the wavelength), or to extract other structural parameters in the nanometer regime. With the SMI microscope presently developed at Kirchhoff Kirchhoff-Institut for physics, this is accomplished by the fact that the illumination intensity is not homogeneous in the object area; in contrast to conventional Epifluoreszenz microscopes is spatially modulated. Two laser beams propagating in opposite directions and interfering in axial direction (see illustration right) are used to set up a standing wave field with intensity modulation along the optical axis. The principle of spatially modulated wavefield has been developed in 1993 by Bailey et al. In the SMI microscopy approach at Heidelberg, the object is moved in highly precise steps through the wave field. From this an increase in the axial size- and distance-resolution is gained.


 


Motivation. An operational area of the SMI microscope is the analysis of biological nano-structures and/or the nuclear structure of cells. Here, size and distance measurements in the nano-regime (<~100nm) are highly important. These investigations are based predominantly on distance measurements of punctiform objects e.g. specific chromosome regions. In combination with new fluorescence labeling methods (co-operation with Institutes of Physical Chemistryl and Anorganic Chemistry of the University of Heidelberg) the SMI microscopy will permit non-contact far field-light-microscopic structure investigations down to the molecular level. Thus, a resolution equivalent will be reached competing with those of the atomic force microscopy (AFM), which optical near field microscopy (Scanning Near field Optical Microscopy, SNOM) and related surface methods. Contrary to these however the method can be used to acquire information on the inside of objects.   

Example of use. Investigations at nuclei of bone marrow stem cells showed relations between the three-dimensional (3D-)structure of the chromosomal configuration and the disease aberations known from chronic myeloid leukaemia. In particular a connection between the distance between the chromosome #9 and #22 and the occurrence of a translocation of the ABL region of chromosome #9 to the BCR region of chromosome #22 is assumed. From these translocations, the so-called Philadelphia-chromosome is fused, whose presence correlates closely with the disease specified above. Within the research group "applied optics and information processing", these investigations represent only one example of medical-biophysical research in which the accurate measurement of the distances between chromosomes and/or between chromosome regions are of great importance(SPDM, spectral precision distance microscopy).

Other examples are  the size measurement of specific gene domains e.g. the p53 gene region. (The p53 gene is of high relevance in tumor biology ;Hildenbrand et al. 2005); size determination of telomeric regions in interphase nuclei (Reymann et al. 2008); or combination with localization microscopy (SPDM to generate 3D images of cellular nanostructures with an optical 3D resolution around 40 - 50 nm (Lemmer et al. 2008).

Further information see list of publications