Fast SLM-based linear and nonlinear structured illumination microscopy
Fluorescent microscopy becomes an essential tool for medical and biological investigations due to its major advantages of allowing for minimally invasive observation and rapid optical imaging. It is also a highly desirable tool to study the three dimensional interior of living specimens at a small scale. However, the resolution of optical systems is fundamentally limited by the diffraction of light, which consequently coins the development of super-resolution imaging methods. Structured illumination microscopy (SIM) is one of the super-resolution techniques. SIM provides a two-fold lateral resolution improvement for those types of samples where the fluorescence emission intensity depends linearly on the intensity of the illumination pattern. The concept of SIM is based on the Moiré effect. A structured illumination pattern is projected into the sample and high spatial frequency components of the biological sample, which are normally above the cut-off frequency of the optical transfer function and therefore lost, are then down-modulated to low spatial frequencies that reside inside the passband of the optical transfer function of the microscope. Typically, a lateral resolution of 100 nm becomes achievable in SIM. SIM is a wide-field technique and thus allows fast acquisition of large fields of view. This work discusses methods to improve the acquisition speed of SIM and to further enhance the resolution beyond the usual factor of two using nonlinear SIM (NL-SIM). Improvement of the acquisition speed is achieved by exploiting the advantages of a ferroelectric spatial light modulator (SLM) which offers fast switching of the illumination pattern, a modern sCMOS camera which provides fast readout and a novel synchronization approach between the different opto-electronical components.