PUB - Publikationen an der Universität Bielefeld

Structured illumination microscopy (SIM) is among the most widely used super-resolution fluorescence microscopy techniques for visualizing the dynamics of cellular organelles, such as mitochondria, the endoplasmic reticulum, or the cytoskeleton. In its most wide-spread implementation, SIM relies on the creation of an interference pattern at the diffraction limit using the coherent addition of laser beams created by a diffraction pattern. Spatial light modulators based on liquid crystal displays allow SIM micro-scopes to run at image rates of up to hundreds of super-resolved images per second. Digital micromirror devices are another natural choice for creating interference-based SIM patterns, but are not used to their fullest potential because of the blazed grating effect. This effect arises due to the fixed angles between which the mirrors can be switched, creating a sawtooth arrangement of mirrors and thus leading to a change in the intensity distribution of the diffracted beams. This results in SIM patterns with varying modulation contrast which are prone to reconstruction artifacts. We have carefully studied the blazed grating effect of DMDs by simulations, varying a range of parameters and compared the simulation results with experiments. This allowed us to identify settings which result in very high modulation contrast across all angles and phases required to generate 2-beam SIM pattern. The use of inexpensive industry-grade CMOS cameras as well as low-cost lasers enabled us to construct a cost-effective, high-speed SIM system. Reconstruction of the super-resolved SIM images is achieved on a recently demonstrated parallel-computing platform, which allowed us to visualize living cells with super-resolution at multiple reconstructed frames per second in real time. We demonstrate the versatility of this new platform by imaging cellular organelle dynamics based on live-cell fluorescent stains as well as with fluorescent protein stained samples.