Supercritical angle fluorescence microscopy

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

SAF detection

The radiation behavior of fluorescent molecules is strongly influenced in close proximity to interfaces between dielectric optical waveguides of different refractive index. In microscopy of biological samples, the fluorescent analyte is usually in an aqueous medium ( the refractive index ) on a cover glass (). The critical angle of the water / glass interface is approximately 61 °. Fluorescent dyes with a distance from the glass surface by more than one wavelength to emit no light above the glass. At shorter distances, particularly in the range up to 300 nm, it comes to the production of SAF, that is, fluorescence emission above the critical angle. Thus, the separate collection of SAF leads to a high specificity of the detection volume for the interface.

Which occurs at large angles SAF emission exceeds the capacity of conventional based on light refraction Linsenssysteme. Full coverage of the SAF emission is collimated by an optical waveguide of the SAF emission by total reflection at its parabolic shell surface to a parallel beam. This developed in the research group of Stefan Seeger detection technique forms the basis of the SAF microscopy, but also of biosensors and detection systems for medical diagnostics.

SAF- microscope

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