Image Scanning Microscopy and Metal Induced Energy Transfer: Enhancing Microscopy Resolution in All Directions

Classical fluorescence microscopy is limited in resolution by the wavelength of light (diffraction limit) restricting lateral resolution to ca. 200 nm, and axial resolution to ca. 500 nm (at typical excitation and emission wavelengths around 500 nm). In image Scanning Microscopy (ISM) the focus of a conventional laser-scanning confocal microscope (LCSM) is scanned over the sample, but instead of recording only the total fluorescence intensity for each scan position, as done in conventional operation of an LCSM, one records a small image of the illuminated region. The result is a four- dimensional stack of data: two dimensions refer to the lateral scan position, and two dimensions to the pixel position on the chip of the image‐recording camera. This set of data can then be used to obtain a super‐resolved image with doubled resolution. A second, completely different approach which aims at achieving nanometer resolution along the optical axis is Metal Induced Energy Transfer or MIET. When placing a fluorescent molecule close to a metal, its fluorescence properties change dramatically. In particular, one observes a strongly modified lifetime of its excited state (Purcell effect). This is due to the efficient electromagnetic coupling of the excited state to surface plasmons in the metal, which is similar to Förster Resonance Energy Transfer (FRET), where the energy of an excited donor molecule is transferred into the excited state of an acceptor molecule. We call this effect metal-induced energy transfer or MIET. The MIET-coupling between an excited emitter and a metal film is strongly dependent on the emitter’s distance from the metal. We have used this effect for mapping the basal membrane of live cells with an axial accuracy of ~3 nm.

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Host: Dr. Thorben Cordes