# Increasing Resolution in Live Cell Microscopy by Structured Illumination (SIM)
## 4 Discussion
This potential can, in principle, even be used for in combination with further super-resolution methods, e.g., STED microscopy where SIM may generate a three-dimensional depletion pattern, permitting axial super-resolution STED with moderate light exposure [31]. Although each SIM image has to be calculated from at least nine individual images, acquisition times are short compared to most other super-resolution techniques based on laser beam scanning or on single-molecule localization (only for LSM 880 with Airy scan, Carl Zeiss Jena, Germany, recording times down to about 50 ms are reported). Our present acquisition time for one super-resolution image (including nine single images) is about 5 s, but this time can be shortened to the millisecond range [30; 32] using either galvano scanners (e.g., Elyra 7, Carl Zeiss Jena, Germany) or a spectral light modulator (SLM). So far, the highest frame rate (255 frames per second) was achieved with Elyra 7 and lattice SIM, a technique creating a three-dimensional point pattern (instead of a linear grid) operated in the so-called \"burst\" mode. This technique [33] has the advantage that for recording individual images the pattern has only to be shifted (not rotated), so the image sequence becomes faster. In addition, the modulation depth of lattice SIM is enhanced in comparison with conventional SIM. Compared to scanning techniques, the SLM does not need any movable components, but when selecting the first orders of diffraction, many higher orders of interference have to be discarded. Therefore, either higher laser powers or longer recording times as compared to those of the scanning SIM are required. However, in all cases super-resolution images must be calculated offline from individual SIM images. It should be emphasized that in comparison to commercial systems, our present SIM setup is not optimized for the highest speed. However, it has the advantage that it uses a conventional inverted microscope and can be easily combined with other microscopy techniques, e.g., light sheet microscopy [5], as well as spectral imaging or fluorescence lifetime imaging microscopy (FLIM). In summary, low recording times in combination with low light exposure and the potential for 3D or deep view microscopy might favor SIM in comparison with other super-resolution methods, and SIM might represent a less light consuming alternative for STED or super-localization microscopy in long-term imaging of cell or tissue dynamics [34; 35]. For an increase in axial resolution close to surfaces (e.g., cell membranes), SIM can be combined with total internal reflection microscopy (TIRFM) if an interference structure is created by two laser beams whose angles of incidence are above the critical angle of total internal reflection [36]. Finally, some indirect methods, e.g., Forster resonance energy transfer [37], may further improve the potential of measuring molecular distances in the nanometer range. SIM experiments and evaluation, V.R. ; setting up of equipment, M.P. ; technical support, M.W. ; organization and writing of this manuscript as well as presentation at LALS 2018, H.S. This project was funded by the Ministry of Science and Arts (MWK) Baden-Wurttemberg. The authors thank [PERSON] and [PERSON] (IPHT Jena) as well as [PERSON] and [PERSON] (Univ. Heidelberg) for their valuable cooperation and [PERSON] (Aalen University) for skillful technical assistance. The authors declare no conflict of interest.