Supercritical Angle Fluorescence

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Supercritical Angle Fluorescence (SAF) (dt .: fluorescence above the critical angle ) is a method of microscopy for the fluorescence spectroscopic investigation of biochemical species and structures ( proteins , DNA , cells , tissue). The separate detection of the fluorescence emitted above the critical angle enables the specific investigation of the contact area of ​​the sample with the support glass ( slide , cover glass ).

SAF detection

SAF emission at the water / glass interface

The radiation behavior of fluorescent molecules is strongly influenced when they are very close to interfaces between dielectric light waveguides with different refractive indices . When microscopy of biological samples is used, the fluorescent analyte is mostly in an aqueous medium (refractive index ) on a cover glass ( ). The critical angle of the water / glass interface is approx. 61 °. Fluorescent dyes with a distance to the glass surface of more than one wavelength do not emit any light above into the glass. At smaller distances, especially in the range up to 300 nm, SAF occurs, i.e. H. for fluorescence emission above the critical angle. Thus, the separate collection of SAF leads to a high specificity of the detection volume for the interface.

The SAF emission occurring at large angles exceeds the capacity of conventional lens systems based on refraction . The complete detection of the SAF emission is collimated with an optical fiber which collimates the SAF emission by total reflection on its parabolic surface to form a parallel bundle of rays. This detection technology developed in Stefan Seeger's research group forms the basis of SAF microscopy, but also that of biosensors and detection systems for medical diagnostics.

SAF microscope

A special microscope objective is used for SAF microscopy, which contains a parabolic light guide and a lens system with a high numerical aperture. The inner lens system is used to excite the sample and collect fluorescence emitted below the critical angle, the outer parabolic light guide for collecting SAF. The separate, simultaneous measurement of the fluorescence emitted above and below enables parallel microscopy with two different resolutions along the optical axis.

Individual evidence

  1. ↑ W. Lukosz, R. Kunz: Light emission by magnetic and electric dipoles close to a plane interface. I. Total radiated power. J. Opt. Soc. At the. 67: 1607-1614 (1977).
  2. ↑ W. Lukosz, R. Kunz: Light emission by magnetic and electric dipoles close to a plane interface. II. Radiation patterns of perpendicular oriented dipoles. J. Opt. Soc. At the. 67: 1615-1619 (1977).
  3. ↑ J. Enderlein, T. Ruckstuhl, S. Seeger: Highly efficient optical detection of surface-generated fluorescence. Appl. Opt. 38, 724-732 (1999).
  4. ↑ T. Ruckstuhl, S. Seeger: Light detecting optical device, US6714297, EP1076823, WO9946596 (1998).
  5. ↑ T. Ruckstuhl, M. Rankl, S. Seeger: Highly efficient biosensing using a supercritical angle fluorescence (SAF) instrument , In: Biosens Bioelectron . 18: 1193-1199 (2003).
  6. ^ A. Krieg, S. Laib, T. Ruckstuhl, M. Rankl, S. Seeger: Fast detection of single nucleotide polymorphisms (SNPs) by primer elongation with monitoring of supercritical-angle fluorescence , In: ChemBioChem 5, 1680–1685 ( 2004).
  7. ↑ T. Ruckstuhl, D. Verdes: Supercritical angle fluorescence (SAF) microscopy, Opt. Express 12, 4246-4254 (2004).
  8. ↑ D. Verdes, T. Ruckstuhl, S. Seeger: Parallel two-channel near- and far-field fluorescence microscopy J. Biomed. Opt. 12, 34012 (2007).

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