# Increasing Resolution in Live Cell Microscopy by Structured Illumination (SIM)
## 4 Discussion  
Increasing the resolution in comparison with the Abbe or Rayleigh criterion is still a challenge in optical microscopy. As reported above, SIM is a promising approach in view of the low light exposure needed for imaging living cells. In the present manuscript an increase of the resolution by a factor of 1.7 (theoretically up to a factor of two) has been well documented for two-dimensional samples, e.g., fluorescent beads or cell monolayers. The gain in resolution is still low in comparison to STED or super-localization microscopy, e.g., STORM, where growth of protein fibrils was recorded with a resolution better than 20 nm [29]. However, those methods require increased light exposure and often are not compatible with live cell microscopy. Microscopy of three-dimensional specimens, e.g., cell multilayers, spheroids or tissue biopsies, is still a challenge for all kinds of super-resolution microscopy. In the case of SIM an increase of resolution up to a factor of two can be theoretically maintained in larger depths of a sample, even if long distance objective lenses of lower numeric aperture are needed. However, images are subject to pronounced light scattering: they appear blurred (see Figure 7), and low contrast interference patterns, e.g., those of a spatial light modulator, are hardly visible. This makes the calculation of high-resolution SIM images very difficult. However, it appears possible to combine structured illumination and light sheet microscopy [5] (in an up-modulation mode) and to suppress scattered light outside the light  
Figure 6: MCF-7 breast cancer cells incubated with the mitochondrial marker R123 (5 \(\upmu\)M, 30 min) wide field image (**a**) and SIM image (**b**) (evaluated by [PERSON], IPHT Jena); excitation wavelength: \(\lambda_{0}\) = 488 nm; detection range: \(\lambda_{0}\geq\) 515 nm; Plan Neofluar 40\(\times\)/1.30 oil immersion objective lens. Figure 7: 3T3 fibroblasts from a multilayer incubated with the mitochondrial marker R123 (5 \(\upmu\)M for 30 min); wide field image (**a**) and SIM image (**b**) in a sample depth \(\Delta z\) = 10 \(\upmu\)m. Excitation wavelength: \(\lambda_{0}\) = 488 nm; detection range: \(\lambda_{0}\geq\) 515 nm; Plan Neofluar 40\(\times\)/1.30 oil immersion objective lens. sheet. In this way, imaging in larger depths of the sample may become possible. Furthermore, precise positioning of the specimen in the axial direction is cogent for imaging 3D samples. This option is not available in many customary microscopes, where only the objective lens, but not the specimen can be moved in the axial direction. Therefore, with a fixed position for the tube lens (Figure 2) the interference pattern of two overlapping beams is only maintained over a small depth of the specimen. However, coupling of the sample to a piezo element on the microscope stage permits moving of the sample in the axial direction and seems to solve this problem. The main characteristics of the present SIM system can be summarized as follows:  
* it is a wide-field method requiring only low light doses (up to about 10 J/cm\({}^{2}\)) which permits keeping cells alive after incubation with a fluorescence marker or transfection with a fluorescent protein [20] and also avoids artefacts due to photobleaching [30];
* it is comparably simple, without any moving components, using a commercially available (inverted) microscope;
* microscope lenses of various magnification and aperture can be used. In the present case a 40\(\times\)/1.30 objective lens permits obtaining a comparably large field of illumination for cell-based experiments, but requires cameras with more than 250 pixels per millimeter (e.g., ProgRes cameras by Jenoptik GmbH or an Orca Flash camera set by Hamamatsu Photonics) to image structures of about 100 nm size; and,
* it has the potential for imaging in larger depths of a sample (\"deep view imaging\").