# Increasing Resolution in Live Cell Microscopy by Structured Illumination (SIM)  
Verena Richter  
Institute of Applied Research, Aalen University, Beethovenstr. 1, 73430 Aalen, Germany;  
[EMAIL_ADDRESS]  
Mathis Piper  
Institute of Applied Research, Aalen University, Beethovenstr. 1, 73430 Aalen, Germany;  
[EMAIL_ADDRESS]  
Michael Wagner  
Institute of Applied Research, Aalen University, Beethovenstr. 1, 73430 Aalen, Germany;  
[EMAIL_ADDRESS]  
Herbert Schneckenburger  
Institute of Applied Research, Aalen University, Beethovenstr. 1, 73430 Aalen, Germany;  
[EMAIL_ADDRESS]  
lower. This favors LSFM for experiments performed over a long time period. Experimental setups for LSFM range from stand-alone microscopes to miniaturized modules, which can be adapted to various kinds of commercial microscopes and combined with further experimental techniques [5]. Only in the last 25 years have methods been described with a resolution below the Abbe criterion. They are summarized under the term \"super-resolution microscopy\", and include:  
* stimulated emission depletion (STED) microscopy [6], where the outer regions of an illuminated spot are depleted by stimulated emission within the focus of a second donut-shaped laser beam upon optical excitation in a laser scanning microscope. While a fluorescent spot may thus be confined to 70 nm or less, the irradiance exceeds that of a conventional fluorescence microscope by a factor of 10\({}^{4}\)-10\({}^{5}\), so at the moment live-cell imaging does not appear to be possible, although various approaches to reduce the light exposure considerably are being pursued. Those methods include reduction of state transition cycles (REScue) [7], adaptive-illumination STED nanoscopy [8] or MINFLUX technology with tracking of single molecules in the center of a donut-shaped laser beam [9];
* super-localization microscopy of single molecules located within a thin illuminated layer of a sample. If a single molecule is detected n times, its localization can be determined with precision, \(\Delta\)x = \(\Delta\)x\({}_{0}\)/\(\surd\)n with \(\Delta\)x\({}_{0}\)\(\approx\) 200 nm resulting from the Rayleigh criterion. Therefore, a precision of localization \(\Delta\)x = 20 nm results when n = 100 and \(\Delta\)x = 10 nm when n = 400. Methods based on super-localization microscopy include stochastic optical reconstruction microscopy (STORM), photoactivation localization microscopy (PALM) and related techniques [10; 11; 12]. However, the irradiance needed for single-molecule experiments is still 500-1000 times higher than in conventional fluorescence microscopy, such that cells are expected to survive only a short time. Furthermore, the resolution in CLSM can be increased up to a factor of two if only part of an Airy disk passes the detection pinhole, or if each Airy disk is detected by a large number of \"infinitely small pinholes\". This becomes possible by using several single-detector elements (Airy scan microscopy) or an appropriate camera (image scanning microscopy [13]) as well as by scanning the surface of the pinhole (rescanning confocal microscopy, RCM) [14]. In addition to various scanning techniques, as well as to single-molecule localization techniques, structured illumination microscopy (SIM) has proven its super-resolution potential [15; 16]. A down-modulation of conventionally non-transferable higher spatial frequencies by a periodically-modulated illumination pattern leads to resolution enhancement up to a factor of two compared to the value given by the Abbe criterion. While illumination with three laser beams creates an interference pattern with high spatial resolution in all three dimensions [17], an interference pattern in two dimensions is generated by illumination with two laser beams, e.g., the first diffraction orders of an optical grating or a spatial light modulator (SLM) [18]. Calculation of a super-resolved image commonly needs 9-15 single images with structured illumination of different phases and rotation angles [17; 18].