Ingo Kleppe

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.

Super-resolution microscopy heads towards 3D dynamics

Abstract Resolving fine details of subcellular structures is key to understanding the organization and function of cellular networks. Recent advances in far-field fluorescence microscopy provide the necessary tools to analyze these structures with resolutions well below the classical diffraction limit in all three dimensions. Technical improvements go hand-in-hand with new versions of switchable fluorophores that allow nonlinear optical effects to be more efficiently used to push the resolution limit down further. High contrast combined with the wide spectrum of available colors currently endow these fluorescence-based super-resolution techniques with the power to study the complexity of subcellular organelles and the relation of their constituting components down to the molecular level and under physiological conditions. In this way, they give us a far better understanding of the assembly of macromolecular complexes and their functions within a cell than has been possible before employing conventional imaging methods. In this review, we give an overview of the technical state-of-the art of these technologies, their fundamental and technical trade-offs, and provide typical application examples in this exciting field.

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.