Inexpensive Enhancement of High-Resolution 3-D Fluorescence Microscopy: FAM Fluorescence Activation Microscopy

Optical setups using two opposing micro-objectives have been propagated to enhance the axial resolution in 3-dimensional fluorescence microscopy using various illumination schemes. However, the coherent superposition of two counter propagating beams creates imaging artifacts, i.e. more or less pronounced side-maxima, which can only be effectively removed by arithmetic op-erations if their intensity is less than 50% of the main maximum.

Fluorescence Activation Microscopy (FAM) is a new alternative 3D fluorescence image technique besides the existing confocal or 2-photon excita-tion microscopy. In FAM, photo activatable dyes are used, so that improved axial resolution is achieved solely by the illumination beams in very much the same way than in 2-photon excitation and in contrast to the regular confocal case. In FAM, however, it is not relevant whether point, line or other structured illumination patterns are used as long as activation and excitation are applied simultaneously, permitting the use of devices such as micro lens arrays, birefringent devices, SLMs, LCD or DMD projectors, LED arrays, holographic pattern generators etc, so FAM enables optical sectioning without necessarily requiring confocal optics or 2-photon excitation. Together with fluorescent proteins such as DRONPA fast 3d life cell imaging also becomes possible. Calculated point spread functions (PSF) suggest that this method may perfect other high resolution imaging techniques such as 4Pi, STED and PALM additionally by introducing an axial resolution in the 70nm range.

Further Information: PDF

DKFZ (German Cancer Research Center, Deutsches Krebsforschungszentrum)
Phone: +49-6221-42 2955

Contact
Dr. Ruth Herzog