# Increasing Resolution in Live Cell Microscopy by Structured Illumination (SIM)
## 3 Results  
Fluorescence images of the polystyrene nano-beads (diameters of 100 nm and 200 nm) are depicted in Figure 3 as conventional wide-field images (a,d), wide-field images with a Wiener filter for noise suppression (b,e), and SIM images (c,f). In Figure 3a,b resolution according to the Abbe criterion is lower, and in Figure 3d,e close to the particle size. In addition, particles appear somehow blurred in Figure 3a,d, and overlapping in Figure 3a,b,d,e, while in Figure 3c,f all particles appear clear and well resolved. Figure 1: Comparison of optical transfer functions (OTFs): Wiener filtered wide-field (**a**) and structured illumination with two interfering laser beams at angles of 0\({}^{\circ}\), 60\({}^{\circ}\) and 120\({}^{\circ}\) (**b**). Figure 2: Experimental setup for SIM: The object ray creates an image of the SLM pattern in the plane of the sample upon illumination by the parallel beam of an argon ion laser (\(\lambda_{0}\) = 488 nm expanded by a telescope to a diameter of 10 mm, not shown); the illumination ray is focused in the aperture plane A of the objective lens and results in two parallel beams interfering in the plane of the sample (TL = tube lens, Obj = microscope objective lens). The inset shows a scheme of the “pizza polarizer” with 6 areas of different polarization. In Figure 4a a fluorescence image of a 3T3 fibroblast incubated with Tubulin TrackerTM Green (250 nM for 20 min) is depicted, and the image shows brightly fluorescent microtubules. In comparison with the wide-field image the SIM image (b) shows an improved resolution, and in combination with the software algorithm described in Reference [22] permits detection of a single cell layer. The improvement of resolution by a factor of about 1.7 is also documented by a line scan with Gaussian fit over 0.8 um (c). Here, the calculated diameters of 190 nm (wide-field microscopy with Wiener filter) as well as 115 nm (SIM) fit the theoretical values of 187 nm and 111 nm, respectively, and confirm previous results obtained with other tubulin markers [23,24]. It should be mentioned that the real diameter of a microtubule is about 30 nm and, therefore, considerably smaller. A further application is presented in Figure 5 for doxorubicin (adriamycin), an anthracycline antibiotic, which has been used as a cytostatic drug in cancer chemotherapy for several decades [25]. The drug is taken up by cells due to passive diffusion through the membrane and finally intercalates in DNA strands, where it causes chromatin condensation and initiates apoptosis [26]. Location in the cell nuclei of MCF-7 breast cancer cells is well documented in Figure 5, but in comparison with wide-field microscopy (b), SIM gives an improved resolution for the nuclear envelope from about 220 nm to 120-150 nm (c) and possibly some information on nuclear architecture upon application of this cytostatic agent. Figure 3: Images of fluorescent nano-beads with a size of 100 nm (upper part) as well as 200 nm (lower part) using wide-field microscopy (**a**,**d**), wide-field microscopy with a Wiener filter (**b**,**e**) or SIM (**c**,**f**); excitation wavelength: \(\lambda_{0}=488\) nm; detection range: \(\lambda_{\mathrm{D}}\geq 515\) nm; Plan-Neofluar 40\(\times\)/1.30 oil immersion objective lens. Fluorescence images of individual MCF-7 breast cancer cells incubated with the mitochondrial marker rhodamine 123 (5 uM for 30 min) are depicted in Figure 6. In both parts of this image a non-fluorescent nucleus is surrounded by mitochondria which appear as long fluorescent rods. While the wide-field image (Figure 6a) shows some overlaying, diffuse, and out-of-focus fluorescence, the image calculated from nine images with structured illumination (SIM shown in Figure 6b) shows fluorescence only from the focal plane with enhanced image quality. However, the resolution cannot be specified, since the mitochondria are larger than the resolution in the microscope, and mitochondrial sub-structures cannot be visualized.