# Increasing Resolution in Live Cell Microscopy by Structured Illumination (SIM)
## 2 Materials and Methods  
Fluorescent polystyrene beads of 100 nm and 200 nm diameter were kindly provided by [PERSON] (Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Germany), and calibration slides were prepared as described in Reference [21]. 3T3 murine fibroblasts were cultivated as monolayers or multilayers on microscope cover slips (without any further substrates) in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, pyruvate and antibiotics at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\). 72 h after seeding cells were incubated for 20 min with Tubulin Tracker(tm) Green at a concentration of 250 nM. MCF-7 human breast cancer cells were cultivated as monolayers on microscope cover slips in supplemented Dulbecco's MEM/Ham F-12 at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\). Cells were incubated with the cytostatic agent doxorubicin (4 \(\mu\)M) for 24 h or with the mitochondrial marker rhodamine 123 (5 \(\mu\)M) for 30 min. In all cases cells were rinsed with Earle's Balanced Salt Solution (EBSS) prior to fluorescence microscopy. An argon ion laser with \(\lambda_{0}\) = 488 nm was used for excitation, and fluorescence was detected in an inverted microscope (Axiovert 200M, Carl Zeiss Jena, Germany) using a Plan-Neofluar 40\(\times\)/1.30 (oil immersion) objective lens, a long pass filter for \(\lambda_{D}\geq\) 515 nm and further optics as described below. As reported earlier [17], SIM in 2 dimensions needs an interference pattern recorded for a minimum of three phases (0, 2\(\pi\)/3, 4\(\pi\)/3) at three rotation angles (e.g., 0\({}^{\circ}\), 60\({}^{\circ}\) and 120\({}^{\circ}\)) in order to obtain an optical transfer function (OTF) of rotational symmetry (Figure 1). Interference patterns of an SLM (ferroelectric LCOS, SXGA-3 DM, Forth Dimension Displays, UK) are created by a computer program, and first orders of diffraction of an argon ion laser beam (\(\lambda_{0}\) = 488 nm) are used for structured illumination, as reported in Reference [18]. Compared to conventional gratings, no movable components are needed; however, the \"grating constant\" of the SLM of about 90 \(\mu\)m results in low diffraction angles and requires a large focal length on the collecting lens L\({}_{1}\) (Figure 2) for geometrical separation of the diffraction orders. The SLM is imaged by a telescope system consisting of the lenses L\({}_{1}\) (focal length f\({}_{1^{\prime}}\) = 500 mm) and L\({}_{2}\) (f\({}_{2^{\prime}}\) = 120 mm). This gives an interference pattern of the first diffraction orders with a grating constant of 10.96 \(\mu\)m in the image plane of the SLM, which is reduced to \(\Lambda\) = 274 nm by the microscope objective lens (40\(\times\)/1.30 oil immersion). The magnification factor of the objective lens includes that of the tube lens (TL) (also depicted in Figure 2). The resolution in the present case can be calculated by summing up the spatial frequencies resulting from the Abbe criterion k\({}_{\text{Abbe}}\) = 2A\({}_{\text{N}}\)/\(\lambda\) = 0.00533 nm\({}^{-1}\) (with \(\lambda\) = 488 nm) and the interference pattern k\({}_{\text{grating}}\) = 1/\(\Lambda\) = 0.00365 nm\({}^{-1}\) as d\({}_{\text{min}}\) = (k\({}_{\text{Abbe}}\) + k\({}_{\text{grating}}\))\({}^{-1}\) = 111 nm. In comparison with the Abbe criterion \(\lambda\)/2A\({}_{\text{N}}\) = 188 nm this is an improvement by a factor of 1.70. Further components needed for structured illumination (Figure 2) are a \(\lambda\)/4 plate, a plate of 6 pinholes (to select the first diffraction order of the SLM for 0\({}^{\circ}\), 60\({}^{\circ}\) and 120\({}^{\circ}\) orientation) and a so-called \"pizza polarizer\" to select an azimuthal polarization in each case. In addition, an aperture of 5-8 mm on the microscope objective lens is required for transmission of the two interfering laser beams.