3D SIM microscope

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Comparison of the resolution of confocal laser scanning (top) and 3D SIM microscopy (bottom). Nuclear pores (anti-NPC, red), nucleus envelope (anti- Lamin B, green), and DNA packed in chromatin ( DAPI , blue) were stained simultaneously in a mouse cell. The scale bar corresponds to 1 µm.

The 3D SIM microscope (engl. 3D structured illumination microscope ) realizes a further developed form of light microscopy , the resolutions beyond that of Ernst Abbe described resolution limit allows. The concept of 3D SIM microscopy was first introduced by Lukosz and Marchand in 1963 and further developed by a team led by Mats GL Gustafsson and John W. Sedat at the University of California, San Francisco . Commercial versions are offered by Applied Precision as "OMX", by Carl Zeiss as "ELYRA S.1 or PS.1", and by Nikon as "N-SIM".

Working principle

3D-SIM microscopy uses spatially modulated structured lighting (mostly in the form of a line grid) for fluorescence excitation. Several images of the sample have to be recorded here, the illumination pattern in the sample having to be shifted from one recorded image to the next. This is repeated for several levels of a 3D volume. An image of the object (with increased resolution) can then be calculated from these stored raw images. The increase in resolution is based on the principle of the moiré effect, with the detected images being interpreted as a superposition of the (known) lighting pattern and the (unknown) object frequencies. In the case of a stripe pattern, the phase position of the pattern is shifted in at least 5 steps over a period. Since stripe patterns are only modulated in one direction, raw images must be recorded for at least three orientations of the pattern. In contrast to 2D-SIM variants, with 3D-SIM raw images are saved for an entire volume and the images of the measured volume are used to calculate a volume with increased resolution. This means that the microscopic resolution can be doubled in all three spatial directions.

power

With the OMX, the achievable resolution ranges from 105  nm when illuminated with light with a wavelength of 405 nm and up to 165 nm at a wavelength of 593 nm. This roughly halves the previous resolution limit of approx. 200 nm. With the help of the 3D SIM -Microscopy was the first time researchers were able to see parts of the cell nucleus shell such as membranes and pores that could not be seen in a conventional light microscope; likewise, previously invisible details could be seen on the surface of chromosomes .

Advantages compared to other methods

Although a much higher resolution can be achieved with the help of electron microscopy, live cell microscopy and multicolored images are not possible with this method. One advantage of 3D SIM microscopy compared to some other new types of light microscopy methods beyond the classic resolution limit is that normal fluorescence microscope preparations can be used.

  • Images of cell nuclei and stages of mitosis recorded with 3D-SIM microscopy.

  • Comparison of confocal microscopy with 3D SIM

  • Cell nucleus in the prophase stage from different sides

  • Two mouse cell nuclei in the prophase stage

  • Mouse cell in telophase

Web links

Individual evidence

  1. ↑ W. Lukosz, M. Marchand: Optical imaging, exceeding the diffraction- related resolution limit . In: Optica Acta . 10, No. 3, 1963, pp. 241-255. doi : 10.1080 / 713817795 .
  2. ^ Carl Zeiss MicroImaging GmbH: Superresolution Structured Illumination Microscopy (SR-SIM). White Paper, Carl Zeiss BioSciences, Jena Location 2010 ( PDF ).
  3. ^ MG Gustafsson, L. Shao, PM Carlton et al : Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination . In: Biophysical Journal . 94, No. 12, June 2008, pp. 4957-4970. doi : 10.1529 / biophysj.107.120345 . PMID 18326650 .
  4. ↑ API DeltaVision OMX . Appliedprecision.com. Retrieved June 23, 2010.
  5. ^ Carl Zeiss MicroImaging GmbH: ELYRA Enter the World of Superresolution. Carl Zeiss BioSciences, Jena Location 2011 ( PDF ).
  6. ↑ Nikon N-SIM ( Memento from March 4, 2016 in the Internet Archive ).
  7. ^ IM Dobbie, E. King, RM Parton, PM Carlton, JW Sedat, JR Swedlow, I. Davis: OMX: A New Platform for Multimodal, Multichannel Wide-Field Imaging . In: Cold Spring Harbor Protocols . August 2011, p. 899-909 , doi : 10.1101 / pdb.top121 , PMID 21807861 .
  8. ↑ L. Schermelleh, PM Carlton, S. Haase et al : Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy . In: Science . 320, No. 5881, June 2008, pp. 1332-1336. doi : 10.1126 / science.1156947 . PMID 18535242 .
  9. ^ Carlton PM: Three-dimensional structured illumination microscopy and its application to chromosome structure . In: Chromosome research: an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology . 16, No. 3, 2008, pp. 351-365. doi : 10.1007 / s10577-008-1231-9 . PMID 18461477 .