Gerhard Krampert

Line scan - structured illumination microscopy super-resolution imaging in thick fluorescent samples

Structured illumination microscopy in thick fluorescent samples is a challenging task. The out-of-focus fluorescence background deteriorates the illumination pattern and the reconstructed images suffer from influence of noise. We present a combination of structured illumination microscopy with line scanning. This technique reduces the out-of-focus fluorescence background, which improves the modulation and the quality of the illumination pattern and therefore facilitates the reconstruction. We present super-resolution, optically sectioned images of a thick fluorescent sample, revealing details of the specimens inner structure.

SIM and PALM: high-resolution microscopy methods and their consequences for cell biology

The diffraction limit in traditional fluorescence microscopy (approximately 200 and 600 nanometers in lateral and axial directions, respectively) has restricted the applications in bio-medical research. However, over the last 10 years various techniques have emerged to overcome this limit. Each of these techniques has its own characteristics that influence its application in biology. This paper will show how two of the techniques, Structured Illumination Microscopy (SIM) and PhotoActivated Localization Microscopy (PALM), complement each other in imaging of biological samples beyond the resolution of classical widefield fluorescence microscopy. As a reference the properties of two well known standard imaging techniques in this field, confocal Laser Scanning Microscopy (LSM) and Total Internal Reflection (TIRF) microscopy, are compared to the properties of the two high resolution techniques. Combined SIM/PALM imaging allows the extremely accurate localization of individual molecules within the context of various fluorescent structures already resolved in 3D with a resolution of up to 100nm using SIM. Such a combined system provides the biologist with an unprecedented view of the sub-cellular organization of life.

SIM and PALM as tools to study protein structural organization, numbers, interaction and dynamics

Lately quite a plethora of concepts have been successfully developed, which take resolution beyond the classical limits of a light microscope. Among these structured illumination microscopy (SIM) and photo activated localization microscopy (PALM) hold the promise to provide biologists with unprecedented insights into sub-cellular organizations. A combination of these methods seems particularly attractive as it allows adapting to the required resolution and enables to map single molecules or molecule ensembles in the context of highly resolved structures. SIM achieves two fold resolution enhancements in both lateral and axial directions, so structures can be highly resolved in 3D. Adapting the structuring to the wavelength opens up the avenue for multi-color staining. Hence the distribution of one protein and its associated structure can be viewed in the context of others. Since all common fluorescent dyes can be used sample preparation is straightforward. Besides the classical approach to obtain highly resolved structures with up to 10 times the classical resolution, the power of PALM lies additionally in its ability to count and observe single molecules. As such clustering of molecules can be studied as well as many molecules tracked simultaneously to study their diffusion. New strategies open up the possibility to obtain resolution enhancement in the axial direction as well. These applications start already to have an impact on our view how a cell is organized and how different proteins contribute to its make-up.